CN102311501A - Fusion protein comprising GLP-1 (Glucagon-Like Peptide) or analog thereof and preparation method as well as application thereof - Google Patents

Fusion protein comprising GLP-1 (Glucagon-Like Peptide) or analog thereof and preparation method as well as application thereof Download PDF

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CN102311501A
CN102311501A CN2010102205671A CN201010220567A CN102311501A CN 102311501 A CN102311501 A CN 102311501A CN 2010102205671 A CN2010102205671 A CN 2010102205671A CN 201010220567 A CN201010220567 A CN 201010220567A CN 102311501 A CN102311501 A CN 102311501A
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polypeptide
gly
cooh
fusion protein
glp
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龚珉
徐为人
郑学敏
任晓文
汤立达
王玉丽
刘巍
孟凡翠
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Tianjin Institute of Pharmaceutical Research Co Ltd
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Tianjin Institute of Pharmaceutical Research Co Ltd
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Abstract

The invention belongs to the field of medicaments for diabetes and obesity, and particularly relates to a recombinant fusion protein with capability of prolonging in-vivo half life of glucagon-like peptide and a preparation method thereof, a medicinal composition comprising the fusion protein and application of the fusion protein to preparation of medicaments for treating the diabetes and/or the obesity. The fusion protein is shown by the following general formula I: A-a-B-a'-A' (I), wherein A and A' are independently polypeptides 1 with amino acid sequences of SEQ ID NO 53 or polypeptides 2 with amino acid sequences of SEQ ID NO 54; B is a polypeptide 3 with amino acid sequences of SEQ ID NO 55 or polypeptide 4 with an amino acid sequence of SEQ ID NO 56; and a and a' independently connecting peptides consisting of 0-5 amino acid residues, such as 0-3 amino acid residues.

Description

Fusion protein containing GLP-1 or analog thereof, preparation method and application thereof
Technical Field
The invention belongs to the field of diabetes-related medicines. In particular, the present invention relates to a fusion protein having an extended half-life in vivo of glucagon-like peptides. The invention also relates to a preparation method of the fusion protein and application of the fusion protein in preparation of diabetes drugs.
Background
The glucagon-like peptide 1 (GLP-1) is mainly a polypeptide consisting of 37 amino acids secreted by L cells of small intestine, and the active forms of the polypeptide are GLP-1(7-37) OH and GLP-1(7-36) NH2(Mojsov S, J Clin invest.1987 Feb; 79 (2): 616-9). GLP-1 can significantly reduce postprandial blood sugar of human, stimulate insulin production, and play a certain weight-reducing effect without causing hypoglycemia (Drucker D J, diabetes, 1998 Feb; 47 (2): 159-69). Recent studies have also shown that GLP-1 has a regenerative effect on the pancreas (Drucker D J, 2003 Dec; 144 (12): 5145-8). However, GLP-1(7-37) has a serum half-life of only 3-5 minutes. In the aspect of therapeutic application of GLP-1 peptide, Exenatide, a compound of human GLP-1 analogue Exendin-4 from lizard saliva, is marketed in 2005 in the United states as a hypoglycemic drug, the half-life period of the compound in blood is about 2.5 hours, and the compound needs to be injected before breakfast and supper every day, so that the administration by multiple injections every day in clinical use is very inconvenient. Thus, extending the in vivo duration of GLP-1 analogs is useful for clinical applicationsHas important significance.
There have been many studies on the solution of the problem of short retention time of GLP-1 analogs in vivo using the GLP-1 analog fusion protein technology (e.g., chinese patent applications CN90101167.3, CN200710018734.2, CN200410054397.9, CN01820232.2, CN200380110152.7, CN200510039265.3, CN200610127237.1 and CN 200910009642.7). However, the prior art is still far from clinically desirable targets.
Disclosure of Invention
Aiming at the defects in the prior art, the fusion protein technology is adopted to overcome the problem of short half-life period of GLP-1. It is an object of the present invention to provide a fusion protein having the activity of GLP-1 or an analog thereof and having a long residence time in vivo, thereby overcoming the prior art disadvantage that GLP-1 analog drugs need to be administered by multiple daily injections. It is another object of the invention to provide a nucleic acid sequence encoding the fusion protein, a vector comprising the nucleic acid sequence and a host cell comprising the vector. It is still another object of the present invention to provide a method for preparing the fusion protein. The invention also aims to provide application of the fusion protein, including application thereof in preparing medicines for treating diabetes and/or obesity. It is a further object of the present invention to provide a pharmaceutical composition comprising the fusion protein.
The technical scheme for realizing the purpose is as follows:
in one aspect, the present invention provides a fusion protein represented by the following general formula I:
A-a-B-a’-A’
(I)
wherein,
A. a' is independently polypeptide 1 with an amino acid sequence of SEQ ID NO 53 or polypeptide 2 with an amino acid sequence of SEQ ID NO 54;
b is polypeptide 3 with an amino acid sequence of SEQ ID NO 55 or polypeptide 4 with an amino acid sequence of SEQ ID NO 56;
a. a' is independently a linker peptide consisting of 0-5 amino acid residues, e.g. 0-3 amino acid residues.
In the above fusion protein, the amino acids constituting the linker peptide may be selected from glycine, alanine, isoleucine and valine; glycine is preferred. Preferably, one of A and A' is N-terminally attached to the linker peptide and the other is C-terminally attached to the linker peptide.
Specifically, the structure of the fusion protein is as follows:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 4-polypeptide 2-COOH;
NH2-polypeptide 1- (GLY)5-polypeptide 4- (GLY)5-polypeptide 1-COOH;
NH2-polypeptide 1- (ALA)2-polypeptide 4- (ILE)3-polypeptide 1-COOH; or
NH2-polypeptide 1-VAL-polypeptide 4- (VAL)4-polypeptide 1-COOH;
preferably, the structure of the fusion protein is as follows:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-polypeptide 2-COOH; or
NH2-polypeptide 2-polypeptide 4-polypeptide 2-COOH;
more preferably, the structure of the fusion protein is as follows:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH; or
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 2-COOH.
Accordingly, the invention also provides a nucleic acid sequence encoding the fusion protein, a vector comprising the nucleic acid sequence, and a host cell comprising the vector.
In another aspect, the present invention also provides a method for producing the above fusion protein, which comprises the steps of transcribing and translating the above nucleic acid sequence under conditions in which a detectable amount of the fusion protein is expressed.
Specifically, the preparation method of the fusion protein comprises the following steps:
1) constructing a nucleic acid sequence according to the fusion protein;
2) constructing an expression vector comprising the nucleic acid sequence constructed in step 1);
3) using the expression vector constructed in step 2) to transfect or transform a host cell and allowing the nucleic acid sequence to be expressed in the host cell;
4) purifying to obtain the fusion protein.
In still another aspect, the present invention provides the use of the above fusion protein, nucleic acid sequence, vector or host cell in the preparation of a medicament for treating diabetes, obesity, and/or diabetes and obesity-related diseases.
In still another aspect, the present invention provides a pharmaceutical composition comprising the above fusion protein and one or more pharmaceutically acceptable excipients. Preferably, the pharmaceutical composition is a liquid injection or a freeze-dried injection.
Compared with the prior art, the invention has at least the following advantages:
1. the fusion protein has long in-vivo retention time, avoids the defect that like products need to be injected and administered for multiple times every day when being used for treatment, and is convenient for clinical application.
2. The fusion protein of the invention is preferably derived from a human native sequence, reducing the risk of potential immunogenicity of the fusion protein in humans.
3. The fusion protein can be concentrated to medicinal concentration, can be used as a medicinal active ingredient to be prepared into a medicinal composition together with one or more pharmaceutically acceptable carriers and other components, and is used for treating diseases such as diabetes, obesity and the like and related diseases or diseases.
The following is a detailed description of the invention:
the invention aims to overcome the defects that clinically GLP-1 or analog medicine has short retention time in vivo, so that the treatment effect is short and multiple times of injection administration are required every day, and provides the fusion protein containing GLP-1 or analog with the structure of the general formula I and the application thereof in preparing the medicine for treating diabetes.
Another purpose of the invention is to provide a pharmaceutical composition, which contains the fusion protein containing GLP-1 or the analog thereof shown in the structure of the general formula I as the effective component, and one or more pharmaceutically acceptable carriers, excipients or diluents and the like.
The present invention will now be described one by one with reference to the purpose of the present invention.
The GLP-1 or analog-containing fusion protein of the invention can also be shown as the following general formula I':
a-linker peptide 1-polypeptide 2-linker peptide 2-A'
I’
Wherein,
A. a' is GLP-1 or Exendin-4 respectively;
polypeptide 2 is human serum albumin HSA or human immunoglobulin Fc;
the connecting peptide 1 and the connecting peptide 2 are respectively 0-5 amino acid residues, and the amino acid residue is preferably one or more of GLY, ALA, ILE and VAL;
a and A' may be linked to other peptides at the N-terminus and C-terminus.
GLP-1-or analogue-containing fusion proteins according to formula I', preferably wherein,
A. a' is GLP-1 or Exendin-4 respectively;
polypeptide 2 is human serum albumin HSA or human immunoglobulin Fc;
the connecting peptide 1 and the connecting peptide 2 are respectively 0-3 amino acid residues, and the amino acid residues are preferably: GLY;
a and A' may be linked to other peptides at the N-terminus and C-terminus.
More preferred GLP-1 or analog-containing fusion proteins of the invention are selected from the group consisting of:
NH2GLP-1-GLY-HSA-GLY-GLP-1COOH
NH2GLP-1-GLY-Fc-GLY-GLP-1COOH
NH2GLP-1-HSA-GLY-GLP-1COOH
NH2GLP-1-Fc-GLY-GLP-1COOH
NH2GLP-1-Fc-GLP-1COOH
NH2GLP-1-HSA-GLP-1COOH
NH2GLP-1-GLY-HSA-GLY-Exendin-4COOH
NH2GLP-1-GLY-Fc-GLY-Exendin-4COOH
NH2GLP-1-HSA-GLY-Exendin-4COOH
NH2GLP-1-Fc-GLY-Exendin-4COOH
NH2Exendin-4-GLY-HSA-GLY-GLP-1COOH
NH2Exendin-4-GLY-Fc-GLY-GLP-1COOH
NH2Exendin-4-HSA-GLY-GLP-1COOH
NH2Exendin-4-Fc-GLY-GLP-1COOH
NH2Exendin-4-GLY-HSA-GLY-Exendin-4COOH
NH2Exendin-4-GLY-Fc-GLY-Exendin-4COOH
NH2Exendin-4-HSA-GLY-Exendin-4COOH
NH2Exendin-4-Fc-GLY-Exendin-4COOH
NH2Exendin-4-HSA-Exendin-4COOH
NH2Exendin-4-Fc-Exendin-4COOH
NH2GLP-1-(GLY)5-Fc-(GLY)5-GLP-1COOH
NH2GLP-1-(ALA)2-Fc-(ILE)3-GLP-1COOH
NH2GLP-1-VAL-FC-(VAL)4-GLP-1COOH
in the aforementioned fusion protein, the amino acid sequence of GLP-1 is:
SEQ ID NO 53：7-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-36
the Exendin-4 sequence is as follows:
SEQ ID NO 54：GEGTFTSDLSKQMEEEAVRLFIEWLKIGGPSSGAPPPS
the HSA amino acid sequence is:
SEQ ID NO 55：
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALV
LIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF
GDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV
RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKA
AFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQKFGER
AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECAD
DRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP
SLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRL
AKTYETTLEKCCAAADPHECYAKVFDEFKLVEEPQNLIKQNCELFE
QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE
AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF
SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK
PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAA
LGL
the IgG Fc amino acid sequence is:
SEQ ID NO 56：
WQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKC
QGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLS
TLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVT
YLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETV
NITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRS
STRDWKDHKFKWRKDPQDK
the corresponding nucleic acid sequence of the fusion protein, or a nucleic acid sequence comprising the nucleic acid sequence, can be obtained based on the amino acid sequence of the fusion protein. The fusion protein can be obtained by means of a conventional technique in the art using its nucleic acid sequence or a sequence comprising its nucleic acid sequence. The nucleic acid and amino acid sequences of the above fusion proteins are published by NCBI and belong to recognized sequences.
The invention provides methods for producing such fusion proteins, which methods may include the step of transcribing and translating the nucleic acid sequence of the above-described fusion protein under conditions in which a detectable amount of the fusion protein is expressed.
In addition, the preparation method of the fusion protein provided by the invention can also comprise the following steps:
1. construction of DNA encoding the heterologous fusion protein of the invention
Wild-type albumin and immunoglobulins in the heterologous fusion proteins of the invention may be obtained from a variety of sources, for example, these proteins may be obtained from a cDNA library from tissues or cells expressing wild-type albumin and mRNA for immunoglobulins. The invention adopts a standard PCR method to screen the mRNA of related heterologous fusion protein, and designs a primer through the DNA and protein sequence of the disclosed albumin and immunoglobulin.
The heterologous fusion proteins (albumin, immunoglobulins) of the invention are preferably derived from human native sequences to reduce the potential immunogenic risk of the fusion protein in humans. In addition, the immunoglobulin in the present invention preferably contains only an Fc fragment of the immunoglobulin. In the invention, PCR primers are designed according to the public sequences of human serum albumin and human immunoglobulin, and the mRNA of the human serum albumin and the human immunoglobulin is extracted from human blood RNA. The heterofused mRNA is reverse transcribed into DNA by standard mRNA reverse transcription techniques.
2. Construction of DNA for GLP-1 or an analog of the present invention:
the invention refers to a synthetic method of the full length of double-stranded DNA in GLP-1 in patent CN1363654A to synthesize the full length of double-stranded DNA of GLP-1, Exendin-4 and other GLP-1 analogues. GLP-1, Exendin-4 and other GLP-1 analogues are subjected to sequence verification through standard DNA sequencing. GLP-1, Exendin-4 and other GLP-1 analogs in the present invention refer to GLP-1, Exendin-4 and other GLP-1 analogs with or without a linking peptide attached.
3. Constructing the GLP-1 heterologous fusion protein carrier of the invention:
GLP-1, Exendin-4 and other GLP-1 analogs with different connecting short peptides are prepared by utilizing the PCR mutation technology which is conventional in the field and are used for connecting with protein expression plasmids. The ligation technique is a routine operation in the art, and the GLP-1 analogue containing the linker peptide is ligated with the protein expression plasmid by T4 ligase at the BamHI and NdeI sites by PCR to obtain an expression vector of all fusion proteins in the invention (schematic diagram is shown in FIG. 3). Once an expression vector encoding the complete fusion protein has been produced, it can be used to infect or transform a host cell, such as E.coli, to produce the fusion protein. Techniques for transforming or transfecting cells with recombinant DNA vectors are routine in the art.
4. General methods for recombinant expression of heterologous fusion proteins of the invention:
host cells are transfected or transformed with the expression vectors of the invention for expression of heterologous fusion proteins. The principles, techniques (e.g., media, temperature, pH, etc.) for increasing Cell culture can be found in Mammalian Cell Biotechnology, referring to art-routine heterologous protein expression techniques to optimize expression of various heterologous fusion proteins of the invention: a Practical Approach, m. The expression of the fusion protein containing GLP-1 and the analog thereof adopts the conventional lac promoter escherichia coli fusion protein expression technology in the field, and IPTG is used for starting the generation of the GLP-1 analog fusion protein.
5. Purification of heterologous fusion proteins of the invention:
once the heterologous fusion protein of the invention is expressed in an appropriate host cell, the fusion protein of the invention can be purified by standard protein isolation and purification techniques. For example, crude purification of the fusion protein is performed based on a tag in the fusion protein expression vector. In the invention, the purified heterologous fusion protein can be concentrated to medicinal concentration by an ultrafiltration method, and the fusion protein is treated by ultraviolet rays to improve the medicinal safety.
6. Characterization of the fusion proteins of the invention:
in the present invention, the fusion protein of the present invention can be characterized in a number of ways. Some of these methods include: SDS-PAGE coupled with protein staining or immunoblotting techniques. All heterologous fusion proteins of the invention are identified by immunoblotting, for example using anti-IgG antibodies, anti-HSA antibodies, anti-GLP-1 antibodies and anti Exendin-4 antibodies. The selection of antibodies in immunoblot characterization of the invention is shown in table 1.
TABLE 1 antibodies used in the Western blotting method to which this patent relates
Figure BSA00000176849900081
Note: a and B are both connecting peptides, and the amino acid sequences are shown in specific examples.
The invention also provides a pharmaceutical composition comprising the fusion protein.
The fusion protein containing GLP-1 and GLP-1 analogues can be prepared into a pharmaceutical composition together with one or more pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise: water-soluble fillers, PH adjusters, stabilizers, water for injection, osmotic pressure adjusters, and the like. The pharmaceutical composition can be administered by intramuscular, intravenous or subcutaneous injection, and the preferred dosage form is lyophilized or solution injection.
The water-soluble filler auxiliary material comprises: mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, etc.
The pH regulator comprises non-volatile acids such as citric acid, phosphoric acid, hydrochloric acid, etc., and physiologically acceptable organic or inorganic acids and bases such as potassium hydroxide, sodium hydroxide, potassium or ammonium, sodium carbonate, potassium or ammonium salt, sodium bicarbonate, potassium or ammonium salt, etc., and salt thereof, and one or more of the above substances;
the stabilizer comprises: EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate or trihydroxymethyl aminomethane or a combination of a plurality of the components. Preferably one or more of sodium pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000 and tris (hydroxymethyl) aminomethane.
The osmotic pressure regulator comprises one or a combination of sodium chloride and potassium chloride.
The present invention provides an injection preparation comprising the fusion protein, and the preparation of the injection preparation can be exemplarily referred to the following methods:
(1) freeze-drying agent
Taking fusion protein containing GLP-1 and GLP-1 analogues, water-soluble filler, stabilizer, osmotic pressure agent, etc., adding appropriate amount of water for injection, adjusting pH to 5-8.5 to dissolve, adding water to scale, adding 0.1-0.5% active carbon, stirring at 10-25 deg.C for 10-20 min, decarbonizing, filtering with microporous membrane to remove bacteria, packaging the filtrate, lyophilizing to obtain white loose block, and sealing.
(2) Solution injection
Taking fusion protein containing GLP-1 and GLP-1 analogues, water-soluble filler, stabilizer, osmotic pressure agent, etc., adding appropriate amount of water for injection, adjusting pH to 5-8.5 to dissolve, adding water to scale, adding 0.1-0.5% active carbon, stirring at 10-25 deg.C for 10-20 min, decarbonizing, filtering with microporous membrane to remove bacteria, packaging the filtrate, and sealing.
The invention has the beneficial effects that: the fusion protein containing GLP-1 and GLP-1 analogues has longer in-vivo retention time, and can be used as an effective component for preparing a medicament for treating diabetes. The effective dosage range is quite broad, and the specific dosage can be determined by the physician according to the relevant circumstances, including: the physical state of the subject, the route of administration, the age, body weight, individual response to the drug, severity of the symptoms, and the like. Generally, an effective amount of the active ingredient of the present invention is 1X 10-7 to 20 mg/kg/day, which may be administered as a single dose or as a dose of the ingredient.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the immunoblot results of the relevant fragments in the fusion protein, wherein 1 is the immunoblot of HSA; 2 is immunoblot of IgG Fc; 3 is an immunoblot of GLP-1; 4 is an immunoblot of Extendin-4.
FIG. 2 shows the effect of fusion proteins from different examples on the glucose tolerance of mice, in particular the hypoglycemic effect of the fusion proteins.
FIG. 3 shows an expression vector of the constructed fusion protein, wherein A represents GLP-1-linking peptide or Exendin-4-linking peptide, B represents Human Serum Albumin (HSA) or human immunoglobulin Fc fragment (IgG Fc), and C represents GLP-1-linking peptide or Exendin-4-linking peptide.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1: NH2GLP-1-GLY-HSA-GLY-GLP-1COOHpreparation of fusion proteins
(1) Vector construction and protein expression purification
The construction of the vector: will be provided withNH2The N-terminal nucleic acid sequence of GLP-1 is constructed and connected with plasmid, and the C-terminal is connected with GLY-HSA sequence. Then, the C-terminus of the resulting vector (plasmid-GLP-GLY-HSA) was ligated to the N-terminus having a GLY-GLP-1 nucleic acid, while the C-terminus was ligated to the plasmid.
NH2DNA sequence required for GLP-1-GLY nucleic acid sequence constructionThe following are listed:
SEQ ID NO 1：
ACGCGGAAGGTACCTTCACCAGCGATGTGAGCAGCTATCTG
SEQ ID NO 2：
TGACCTTCCAGATAGCTGCTCACATCGCTGGTGAAGGTACCTTCC
GCGTG
SEQ ID NO 3：
GAAGGTCAGGCGGCGAAAGAATTTATCGCGTGGCTGGTGAAAGG
TCGTGGC
SEQ ID NO 4：
GCCACGACCTTTCACCAGCCACGCGATAAATTCTTTCGCCGCC
GLY-GLP-1COOHthe DNA sequences required for the construction of the nucleic acid sequences are as follows:
SEQ ID NO 5：
GGCACGCGGAAGGTACCTTCACCAGCGATGTGAGCAGCTATCTG
SEQ ID NO 6：
TGACCTTCCAGATAGCTGCTCACATCGCTGGTGAAGGTACCTTCC
GCGTGGCC
SEQ ID NO 7：
GAAGGTCAGGCGGCGAAAGAATTTATCGCGTGGCTGGTGAAAGG
TCGTGGC
SEQ ID NO 8：
ACGACCTTTCACCAGCCACGCGATAAATTCTTTCGCCGCC
reference is made to the method described in patent CN1363654A, i.e. cooling to form two double-stranded fragments. The two fragments were ligated using T4 ligaseIs GLP-1 full-length sequence. The method specifically comprises the following steps: formed by SEQ ID NO 1, 2, 3, 4 areNH2A GLP-1-GLY nucleic acid sequence; formed by SEQ ID NO5, 6, 7 and 8 is GLY-GLP-1COOHNucleic acid sequences (4 ml each of the double-stranded DNA obtained from SEQ ID 1, 2, 3, 4, 1ml of T4 ligase, 1ml of T4 ligase buffer, 16 ℃ overnight. 1% DNA agarose electrophoresis was used to examine the ligation results, the same procedure was used for the double-stranded DNA obtained from SEQ ID 5, 6, 7, 8).
The invention uses the conventional gene recombination connection technology to prepare the gene by a one-step connection methodNH2GLP-1-GLY-HSA-GLY-GLP-1COOHThe nucleic acid sequence of (1), namely, a sequence having GLY at the N-and C-termini (GLY-HSA-GLY) in PCR amplification of HSA (human total RNA extracted from blood using the RNA Isolation Kit available from Invitrogen, followed by Reverse Transcription of RNA using the Reverse Transcription Kit available from Invitrogen). Finally synthesized by the conventional gene recombination technology in the fieldNH2GLP-1-GLY-HSA-GLY-GLP-1COOHA nucleic acid sequence. The sequence of the invention is verified by DNA sequencing. Similarly, the DNA sequence of the GLP-1 fusion protein obtained by the above method had the cleavage sites [ NdeI and BamHI ] at the N-and C-termini used for ligation with E.coli protein expression vector pET28C]. The method of the present invention is characterized by the restriction enzyme endonuclease technology and T4 ligase ligation technology which are conventional in the artNH2GLP-1-GLY-HSA-GLY-GLP-1COOHThe nucleic acid sequence was ligated with the E.coli expression plasmid pET28 c.
The constructed pET28cNH2GLP-1GLY-HSA-GLY-GLP-1COOHThe cells were transformed into BL21(DE3) competent cells by heat shock transformation, which is a technique conventional in the art. The transformed fusion protein expression vector was cultured on LB plates containing kanamycin antibiotic to select cells having kanamycin resistance. After the obtained bacterial cells with kanamycin resistance are subjected to amplification culture, plasmid extraction and purification are carried out, and the sequence identification of the plasmid is completely correct.
The overnight broth was diluted 1: 100 in LB fresh medium, incubated at 37 ℃ until OD600 reached 0.6, expression of the fusion protein was initiated using IPTG, and the system temperature was reduced to 25 ℃.
After the fusion protein is expressed for 16h, centrifuging the bacterial liquid, collecting, and ultrasonically crushing thalli, wherein the operation procedure is as follows: 10cycles, 15 sec/cycle. And (4) centrifuging the crushed bacteria liquid at a high speed, and injecting the supernatant into a nickel column. The fusion protein containing the 6 × HIS tag was captured with a nickel column and eluted using a concentration gradient of imidazole. The eluted proteins were collected and dialyzed and concentrated using a centrifuge tube with a 10kDa semipermeable membrane. The purified fusion protein was cleaved by thrombin treatment to remove the 6 × HIS tag at the N-terminus, and the cleavage technique of thrombin was referred to the experimental manual of Invitrogen corporation. And separating the reaction mixture by using a GelFilation S-200 protein purification column, and simultaneously purifying the target fusion protein. Filtering and concentrating the purified fusion protein by a 10K filter membrane, and performing ultraviolet sterilization treatment.
(2)NH2GLP-1-GLY-HSA-GLY-GLP-1COOHCharacterization of the fusion protein
Immunoblot identification of human serum albumin fusion proteins
The purified HSA fusion protein was diluted in PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse anti-human HSA monoclonal antibody (Santa Cruz, USA) at room temperature for 1 hour, followed by secondary antibody binding with HRP-labeled rabbit anti-mouse polyclonal antibody and luminescent development with ECL kit (Amersham Pharmacia Biotech, USA). Results the fusion protein of the invention was better expressed as shown in figure 1 by immunoblotting for HSA.
Immunoblot identification of GLP-1 fusion proteins
The purified protein will also be identified for GLP-1 expression using a corresponding immunoblotting procedure. The purified fusion protein was diluted with PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse anti-human GLP-1 monoclonal antibody (Santa Cruz, USA) at room temperature for 1 hour, followed by secondary antibody binding with HRP-labeled rabbit anti-mouse polyclonal antibody and luminescent development with ECL kit (Amersham Pharmacia Biotech, USA). The results are shown in figure 1 for GLP-1 immunoblot results showing that the fusion protein of the invention is better expressed.
Example 2: NH2GLP-1-GLY-IgG Fc-GLY-GLP-1COOHpreparation of fusion proteins
(1) Construction of vector and protein expression and purification
With reference to the procedure of example 1, from SEQ ID NO 1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO 4 and SEQ ID NO5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 in example 1 were prepared respectivelyNH2GLP-1-GLY and GLY-GLP-1COOHAnd (3) fragment. Extraction was performed based on the conventional IgG Fc DNA sequence, and the N-and C-segments of the fished IgG Fc were made to have GLY sequence by PCR mutagenesis (to give GLY-IgG Fc-GLY). Connecting GLY-IgG Fc-GLY with GLP-GLY and GLY-GLP to obtainNH2GLP-1-GLY-IgGFc-GLY-GLP-1COOH. According to the contents of reference example 1, the process was carried outNH2GLP-1-GLY-IgG Fc-GLY-GLP-1COOHAnd (3) expression and purification of the fusion protein.
(2) Characterization of the fusion protein
Immunoblot identification of human immunoglobulin fusion proteins
The purified human immunoglobulin fusion protein was diluted in PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse anti-human IgG Fc monoclonal antibody (Santa Cruz, USA) at room temperature for 1 hour, followed by secondary antibody binding with HRP-labeled rabbit anti-mouse antibody and luminescent development with ECL kit (Amersham Pharmacia Biotech, USA). As a result, the fusion protein of the present invention was well expressed as shown in FIG. 1, which is the result of immunoblotting for IgG.
Immunoblot identification of GLP-1
In the invention, the purified protein is also used for identifying the expression of GLP-1 by adopting a corresponding immunoblotting method. The purified fusion protein was diluted with PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse anti-human GLP-1 monoclonal antibody (Santa Cruz, USA) at room temperature for 1 hour, followed by secondary antibody binding with HRP-labeled rabbit anti-mouse polyclonal antibody and luminescent development with ECL kit (Amersham Pharmacia Biotech, USA).
Example 3: NH2GLP-1-HSA-GLY-GLP-1COOHpreparation of fusion proteins
In this example, the connecting peptide GLY was linked only to the C-terminus of HSA, but it was only necessary to prepare a peptide having no GLY at the C-terminus in this exampleNH2GLP-1 sequence. Preparation ofNH2GLP-1 is expressed by the following sequence (SEQ ID NO 9, SEQ ID NO 10). At the same time, HSA was fished without GLY at its N-terminus.
SEQ ID NO 9：
GAAGGTCAGGCGGCGAAAGAATTTATCGCGTGGCTGGTGAAAGG
TCGT
SEQ ID NO 10：
ACGACCTTTCACCAGCCACGCGATAAATTCTTTCGCCGCC
In this example, SEQ ID NO 9 and SEQ ID NO 10 were substituted for SEQ ID NO3 and SEQ ID NO 4 in example 1, and synthesized by the procedure described in example 1NH2GLP-1 and GLY-GLP-1COOH. Prepared using the method of example 1NH2GLP-1-HSA-GLY-GLP-1COOHAnd ligated with the pET28c plasmid. After the expression vector of the fusion protein in this example was constructed, the fusion protein in this example was expressed, purified and identified according to the method in example 1.
Example 4: NH2GLP-1-IgG Fc-GLY-GLP-1COOHpreparation of fusion proteins
Operation, preparation of reference example 3NH2GLP-1 and GLY-GLP-1COOH. The procedure of reference example 2 fished DNA for IgG Fc while leaving the N-and C-termini free of GLY.
The fusion protein in this example was expression-purified and identified by reference to the protein expression, purification and identification method in example 2.
Example 5: NH2GLP-1-HSA-GLP-1COOHpreparation of fusion proteins
In this example, HSA was directly linked to GLP-1 at the N-terminus and C-terminus, respectively, and therefore, in this example, it was necessary to prepare a peptide not containing a linking peptideNH2GLP-1 and GLP-1COOH。NH2GLP-1 was prepared according to the method of example 3. Preparation of GLP-1COOHThe DNA sequences used were as follows:
SEQ ID NO 11：
ACGCGGAAGGTACCTTCACCAGCGATGTGAGCAGCTATCTG
SEQ ID NO 12：
TGACCTTCCAGATAGCTGCTCACATCGCTGGTGAAGGTACCTTCC
GCGTG
the substitution of SEQ ID NO5, SEQ ID NO 6 in example 1 by SEQ ID NO 11, SEQ ID NO 12 as described above. SEQ ID NO 9, SEQ ID NO 10 substituted for SEQ ID NO3, SEQ ID NO 4 in example 1.
Prepared by the method of reference example 1NH2GLP-1-HSA-GLP-1COOHWas ligated with the expression vector plasmid pET28c and transformed into E.coli BL21(DE3) bacteria. The methods of expression, purification and identification of the fusion proteins were all in accordance with the method described in example 1.
Example 6: NH2GLP-1-IgG Fc-GLP-1COOHpreparation of fusion proteins
Operation of reference example 5, preparationNH2GLP-1 and GLP-1COOH. The IgG Fc hook in this example did not contain linker peptide.
The fusion protein in this example was purified and identified by expression according to the nucleic acid ligation, protein expression, purification and identification method in reference example 2.
Example 7: NH2GLP-1-GLY-HSA-GLY-Exendin-4COOHpreparation of fusion proteins
(1) Vector construction and protein expression, purification
NH2Construction of GLP-1-GLY synthesized by the method in reference example 1 of SEQ ID NO 1, SEQ ID NO2, SEQ ID NO3 and SEQ ID NO 4. GLY-Exendin-4COOHThe construction of the double-stranded DNA of SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16 reference example 1 was performed using the following sequences:
SEQ ID NO 13：
GGCCACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGAT
GGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 14：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTGCCG
SEQ ID NO 15：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 16：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared separately by the procedure described in reference example 1NH2GLP-1-GLY，GLY-Exendin-4COOHAnd GLY-HSA-GLY, and expression and purification thereofNH2GLP-1-GLY-HSA-GLY-Exendin-4COOHA fusion protein.
(2) Characterization of the fusion protein
Immunoblot identification of human serum albumin fusion proteins
The purified HSA fusion protein was diluted in PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse anti-human HSA monoclonal antibody (Santa Cruz, USA) at room temperature for 1 hour, followed by secondary antibody binding with HRP-labeled rabbit anti-mouse polyclonal antibody and luminescent development with ECL kit (Amersham Pharmacia Biotech, USA). The results are shown in FIG. 1 for the immunoblotting of Exendin-4, which shows that the fusion protein of the present invention is well expressed.
Immunoblot identification of GLP-1 and Exendin-4
In the invention, the purified protein is also used for identifying the expression of GLP-1 and Exendin-4 by adopting a corresponding immunoblotting method. The purified fusion protein was diluted with PBS containing 1% SDS and 2mg bromophenol blue. After boiling for denaturation, 20. mu.l of the sample was placed on SDS-PAGA gel, and after electrophoresis, the protein was transferred to a nitrocellulose membrane. The cells were closed in a greenhouse with 5% skim milk for 2 hours, and incubated with mouse monoclonal antibodies against human GLP-1 and Exendin-4 (Santa Cruz, USA) at room temperature for 1 hour, and then secondary antibody binding was performed with HRP-labeled rabbit polyclonal antibody against mouse, followed by luminescence development with ECL kit (Amersham pharmacia Biotech, USA).
Example 8: NH2GLP-1-GLY-IgG Fc-GLY-Exendin-4COOHpreparation of fusion proteins
With reference to the procedure of example 7, respective preparationsNH2GLP-1-GLY and GLY-Exendin-4COOHAnd GLY-IgG Fc-GLY was prepared by the method described in reference example 2.
Reference example 1 operation, expression and purificationNH2GLP-1-GLY-IgG Fc-GLY-Exendin-4COOHA fusion protein.
Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 9: NH2GLP-1-HSA-GLY-Exendin-4COOHpreparation of fusion proteins
Without linker peptide as described in reference example 5NH2Preparation method of GLP-1 synthesized in the exampleNH2GLP-1, and with reference to GLY-Exendin-4 described in example 7COOHThe preparation method synthesizes the needed GLY-Exendin-4COOH. The present example was carried out by referring to the gene recombination and fusion protein expression purification techniques described in example 1NH2GLP-1-HSA-GLY-Exendin-4COOHAnd (3) preparing the fusion protein.
Protein characterization was performed for HSA and GLP-1, according to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7.
Example 10: NH2GLP-1-IgG Fc-GLY-Exendin-4COOH
reference example 9, wherein the fusion protein is replaced by human immunoglobulin.
Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 11: NH2Exendin-4-GLY-HSA-GLY-GLP-1COOHpreparation of fusion proteins
NH2The DNA sequence required for the preparation of Exendin-4-GLY is as follows:
SEQ ID NO 17：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 18：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 19：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGCGGC
SEQ ID NO 20：
GCCGCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCA
GCCATTCGATAAA
GLY-GLP-1COOHwas prepared by the method of reference example 1. And prepared by reference to the procedure described in example 1NH2Exendin-4-GLY-HSA-GLY-GLP-1COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
Protein characterization was performed for HSA and GLP-1, according to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7.
Example 12: NH2Exendin-4-GLY-IgG Fc-GLY-GLP-1COOHpreparation of fusion proteins
Reference example 11, but the fusion protein in this example can be replaced by human immunoglobulin.
Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 13: NH2Exendin-4-HSA-GLY-GLP-1COOHpreparation of fusion proteins
Having no linker peptide at the C-terminusNH2The DNA sequence required for the preparation of Exendin-4 is:
SEQ ID NO 21：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 22：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 23：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 24：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
GLY-GLP-1COOHwas carried out according to the synthesis method in example 1. Prepared by the method described in reference example 1NH2Exendin-4-HSA-GLY-GLP-1COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
Protein characterization was performed for HSA and GLP-1, according to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7.
Example 14: NH2Exendin-4-IgG Fc-GLY-GLP-1COOHpreparation of fusion proteins
Reference example 13 operation, but the fusion protein in this example is replaced by human immunoglobulin.
Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 15: NH2Exendin-4-GLY-HSA-GLY-Exendin-4COOHpreparation of fusion proteins
GLY-Exendin-4COOHThe constructs of SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16 were synthesized by the method of reference example 1 using the following sequences:
SEQ ID NO 13：
GGCCACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGAT
GGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 14：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTGCCG
SEQ ID NO 15：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 16：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
NH2the DNA sequence required for the preparation of Exendin-4-GLY is as follows:
SEQ ID NO 17：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 18：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 19：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGCGGC
SEQ ID NO 20：
GCCGCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCA
GCCATTCGATAAA
prepared by the method described in reference example 1NH2Exendin-4-GLY-HSA-GLY-Exendin-4COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
With reference to the procedure in example 1, characterization of the protein was performed for HSA. Exendin-4 protein characterization was performed with reference to example 7.
Example 16: NH2Exendin-4-GLY-IgG Fc-GLY-Exendin-4COOHpreparation of fusion proteins
Reference example 16 operation, but the fusion protein in this example is replaced by human immunoglobulin.
Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 17: NH2Exendin-4-HSA-GLY-Exendin-4COOHpreparation of fusion proteins
Having no linker peptide at the C-terminusNH2The DNA sequence required for the preparation of Exendin-4 is:
SEQ ID NO 21：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 22：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 23：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 24：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
GLY-Exendin-4COOHpreparation ofThe DNA sequences required are:
SEQ ID NO 13：
GGCCACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGAT
GGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 14：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTGCCG
SEQ ID NO 15：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 16：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared by the method described in reference example 1NH2Exendin-4-HSA-GLY-Exendin-4COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
With reference to the procedure in example 1, characterization of the protein was performed for HSA. Exendin-4 protein characterization was performed with reference to example 7.
Example 18: NH2Exendin-4-IgG Fc-GLY-Exendin-4COOHpreparation of fusion proteins
Reference is made to the procedure of example 17, but the fusion protein in this example is replaced by a human immunoglobulin.
Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 19: NH2Exendin-4-HSA-Exendin-4COOHpreparation of fusion proteins
NH2The DNA sequence required for the preparation of Exendin-4 is:
SEQ ID NO 21：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 22：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 23：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 24：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
exendin-4 without connecting peptide at N-terminalCOOHThe DNA sequence required for construction is:
SEQ ID NO 25：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 26：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 27：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 28：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared by the method described in reference example 1NH2Exendin-4-HSA-Exendin-4COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
With reference to the procedure in example 1, characterization of the protein was performed for HSA. Exendin-4 protein characterization was performed with reference to example 7.
Example 20: NH2Exendin-4-IgG Fc-Exendin-4COOHpreparation of fusion proteins
Reference example 19 operation, but the fusion protein in this example is replaced by human immunoglobulin.
Exendin-4 protein characterization was performed with reference to example 7. Protein characterization was performed on IgG Fc with reference to example 2.
Example 21: NH2GLP-1-(GLY)5-IgG Fc-(GLY)5-GLP-1COOHpreparation of fusion proteins
NH2GLP-1-(GLY)5The DNA sequences required for the preparation of (A) are:
SEQ ID NO 29：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 30：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 31：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGCGGCGGCGGCGGCGGC
SEQ ID NO 32：
GCCGCCGCCGCCGCCGCTAGGAGGAGGCGCGCCCGACGAAGGGC
CGCCAATTTTCAGCCATTCGATAAA
(GLY)5-GLP-1COOHthe DNA sequences required for the preparation of (A) are:
SEQ ID NO 33：
GGCGGCGGCGGCGGCCACGGCGAAGGCACCTTTACCAGCGATCT
GAGCAAACAGATGGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 34：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTGCCGCCGCCGCCGCCG
SEQ ID NO 35：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 36：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared by the method described in reference example 1NH2GLP-1-(GLY)5-IgG Fc-(GLY)5-GLP-1COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1.
Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Protein characterization was performed on IgG Fc with reference to example 2.
Example 22: NH2GLP-1-(ALA)2-IgG Fc-(ILE)3-GLP-1COOHpreparation of fusion proteins
NH2GLP-1-(ALA)2The DNA sequence required for preparation is:
SEQ ID NO 37：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 38：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCGCTG
GTAAAGGTGCCTTCGCCGTG
SEQ ID NO 39：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGCACGACG
SEQ ID NO 40：
CGTCGTGCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTT
TCAGCCATTCGATAAA
(ILE)3-GLP-1COOHthe DNA sequences required for the preparation of (A) are:
SEQ ID NO 41：
ATCATCATCCACGGCGAAGGCACCTTTACCAGCGATCTGAGCAA
ACAGATGGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 42：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCGCTGGT
AAAGGTGCCTTCGCCGTGGATGATGAT
SEQ ID NO 43：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 44：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared by the method described in reference example 1NH2GLP-1-(ALA)2-IgG Fc-(ILE)3-GLP-1COOHDNA sequence of the fusion protein. The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1. Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Protein characterization was performed on IgG Fc with reference to example 2.
Example 23: NH2GLP-1-VAL-IgG Fc-(VAL)4-GLP-1COOHpreparation of fusion proteins
NH2The DNA sequence required for GLP-1-VAL preparation is as follows:
SEQ ID NO 45：
CACGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGA
AGAAGAAGCGGTTAGGCT
SEQ ID NO 46：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTG
SEQ ID NO 47：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGCGAT
SEQ ID NO 48：
ATCGCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCA
GCCATTCGATAAA
(VAL)4-GLP-1COOHthe DNA sequences required for the preparation of (A) are:
SEQ ID NO 49：
GATGATGATGATCACGGCGAAGGCACCTTTACCAGCGATCTGAG
CAAACAGATGGAAGAAGAAGCGGTTAGGCT
SEQ ID NO 50：
GATAAACAGCCTAACCGCTTCTTCTTCCATCTGTTTGCTCAGATCG
CTGGTAAAGGTGCCTTCGCCGTGATCATCATCATC
SEQ ID NO 51：
GTTTATCGAATGGCTGAAAATTGGCGGCCCTTCGTCGGGCGCGCC
TCCTCCTAGC
SEQ ID NO 52：
GCTAGGAGGAGGCGCGCCCGACGAAGGGCCGCCAATTTTCAGCC
ATTCGATAAA
prepared by the method described in reference example 1NH2GLP-1-VAL-IgG Fc-(VAL)4-GLP-1COOHDNA sequence of the fusion protein.The fusion protein in this example was expressed and purified by referring to the method for expression and purification of the fusion protein in example 1. Protein characterization was performed on GLP-1 by reference to the procedure in example 1. Protein characterization was performed on IgG Fc with reference to example 2.
Example 24:preparation of freeze-dried preparation of pharmaceutical composition containing fusion protein
10mg of the fusion protein of example 1, 0.2g of arginine as a stabilizer and 60000.05g of polyethylene glycol were placed in a container, 80ml of water for injection was added, the mixture was stirred to dissolve the fusion protein, 8g of mannitol and 2g of sorbitol were added, the mixture was stirred to dissolve the fusion protein, the pH was adjusted to 6.0 to 8.0 with 0.1mol/L of sodium hydroxide, and water was added to 100 ml. Adding 0.3g of activated carbon, stirring at 10-25 deg.C for 20min, decarbonizing, filtering with microporous membrane for sterilization, and packaging the filtrate at a volume of 1ml per bottle. Pre-freezing for 2 hr, drying under reduced pressure for 18 hr until the temperature of the sample reaches 15-20 deg.C, drying for 5 hr to obtain white loose block, and sealing to obtain white lyophilized powder for injection (specification of 100 ug/count).
Example 25:preparation of freeze-dried preparation of pharmaceutical composition containing fusion protein
Taking 25mg of the fusion protein in the example 5, adding 0.05g of stabilizer dipotassium hydrogen phosphate and 0.9g of sodium chloride, placing the fusion protein in a container, adding 70ml of water for injection, stirring the mixture to dissolve the fusion protein, adding 12g of mannitol and 7g of lactose, stirring the mixture to dissolve the lactose, adjusting the pH to 6.5-8.5 by using 0.1mol/L of sodium hydroxide, adding water to 100ml, adding 0.25g of activated carbon, stirring the mixture for 20 minutes at 10-20 ℃, decarburizing the mixture, filtering and sterilizing the mixture by using a microporous filter membrane, subpackaging the filtrate by 2ml per branch, pre-freezing the mixture for 2 hours, drying the mixture for 15 hours under reduced pressure after freezing the mixture until the temperature of the sample reaches 15-20 ℃, drying the mixture for 5 hours again to prepare a white loose block, and sealing the sample to obtain a white freeze-dried powder injection with.
Example 26:preparation of pharmaceutical composition solution injection preparation containing fusion protein
Taking 50mg of the fusion protein in the embodiment 19, adding 0.2g of stabilizer tris (hydroxymethyl) aminomethane, 0.1g of sodium metabisulfite and 0.9g of sodium chloride into a container, adding 80ml of water for injection, stirring and dissolving, adding sodium bicarbonate to adjust the pH to be 6-8, adding water to 100ml, adding 0.2g of activated carbon, stirring and adsorbing at 10-25 ℃ for 20 minutes, removing carbon, filtering and sterilizing by adopting a microporous filter membrane, and filling and sealing 2ml of each fusion protein to obtain the fusion protein injection with the specification of 1000 ug/piece.
Example 27:preparation of pharmaceutical composition solution injection preparation containing fusion protein
5mg of the fusion protein of example 22, 0.01g of glutamic acid as a stabilizer, and 0.09g of potassium chloride were put into a vessel, 70ml of water for injection was added thereto and dissolved by stirring, and 2g of glucose and 1g of galactose were added thereto and dissolved by stirring. Adjusting pH to 7.0-8.5 with sodium hydroxide, adding water to 100ml, adding 0.05g of active carbon, stirring and adsorbing at 10-25 deg.C for 20min, removing carbon, filtering with microporous membrane for sterilization, and bottling with 1ml per tube to obtain fusion protein injection with specification of 50 ug/tube.
Example 28:in vivo pharmacokinetics of fusion proteins
GLP-1, Exendin-4 and fusion protein (0.5 mg/kg when GLP-1 or Exendin-4 is contained) are injected into a rat, about 0.2ml of blood is taken from the veins of the ocular plexus before injection and after injection for 0.5, 1, 3, 6, 9, 12, 24, 36 and 48 hours respectively, and serum is prepared for standby.
The GLP-1 concentration measuring method comprises the following steps: the concentration of the fusion protein in the serum of the mouse is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the operation is as follows: plasma was centrifuged at 4 ℃. The concentration of GLP-1 fusion protein in the plasma of mice was determined using a goat anti-mouse GLP-1 concentration assay kit (R & D System Co., Ltd.). The diluted 1: 1 rat plasma samples were added to a 96-well plate, and after the same volume of GLP-1 antibody was added, the plate was incubated at 37 ℃ for 1 hour. Wash the 96 well plate 3 times with wash solution and incubate for 30 min after HRP addition. In the same manner, the 96-well plate was washed, then 50. mu.l of substrate A and substrate B were added, and incubated at 37 ℃ for 10 minutes. The value of 450-570nm is measured after the reaction is stopped by sulfuric acid, and compared with GLP-1 with standard concentration, and pharmacokinetic parameters are calculated according to the ELISA result.
The method for measuring the concentration of Exendin-4 comprises the following steps: the concentration of the fusion protein in the serum of the mouse is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the operation is as follows: plasma was centrifuged at 4 ℃. The concentration of Exendin-4 fusion protein in the plasma of mice was determined using Exendin-4EIA kit (Phoenix Pharmaceuticals, INC). The diluted mouse plasma samples were added to a 96-well plate (50. mu.l), and after adding 25. mu.l of goat anti-mouse primary antibody to Exendin-4, they were incubated at 37 ℃ for 2 hours. The 96-well plate was washed 4 times with wash solution and incubated for 1 hour after addition of 100. mu.l SA-HRP. The 96-well plate was washed in the same manner, and then 100. mu.l of TMB was added and incubated at 37 ℃ for 1 hour. The OD450 values were determined within 20 minutes after termination of the reaction with 2N hydrochloric acid, compared with Exendin-4 of standard concentration and pharmacokinetic parameters were calculated from the ELISA results.
The results of the pharmacokinetics in vivo of the fusion protein are shown in Table 2, the results show that the half-life period of the fusion protein in vivo is obviously prolonged compared with that of GLP-1 and Exendin-4 which are independent, the fusion protein has long-acting property, and the literature reports that the half-life period in vivo of the GLP-1 is only 9 minutes according to the literature.
TABLE 2 end-phase half-life of fusion proteins in rats
Sample source Half-life (hours)
GLP-1 0.1
Exendin-4 2.7
Example 1 5.2
Example 2 6.8
Example 4 8.3
Example 11 15.5
Example 15 13.2
Example 16 16.1
Example 29:effect of fusion proteins on glucose tolerance in rats
Glucose tolerance test in rats given the fusion protein: mice (C57BL/6) fasted overnight (16-18h) were injected subcutaneously with different fusion proteins at a dose of 80. mu.g/kg in terms of GLP-1 or Exendin-4), blank and model control groups were injected with saline, the blank group was not given sugar after 30 minutes and 24 hours of injection, and the other groups were intraperitoneally injected with glucose at 3g/kg body weight and blood samples were taken from the tail tip 15, 30, 60, 90, and 120 minutes after injection, and the blood glucose level was measured with a glucometer and evaluated as an index of the area under the curve (AUC) of blood glucose after sugar administration. The glucose tolerance results of mice given GLP-1 fusion protein are shown in figure 2, and after the fusion protein provided by the invention is given, the glucose tolerance capability of the mice is obviously enhanced, and the effect is maintained for more than 24 hours.
Figure ISA00000176870100011
Figure ISA00000176870100021
Figure ISA00000176870100031
Figure ISA00000176870100051
Figure ISA00000176870100061
Figure ISA00000176870100071
Figure ISA00000176870100081
Figure ISA00000176870100091
Figure ISA00000176870100101
Figure ISA00000176870100111

Claims (12)

1. A fusion protein represented by the following general formula I:
A-a-B-a’-A’
(I)
wherein,
A. a' is independently polypeptide 1 with an amino acid sequence of SEQ ID NO 53 or polypeptide 2 with an amino acid sequence of SEQ ID NO 54;
b is polypeptide 3 with an amino acid sequence of SEQ ID NO 55 or polypeptide 4 with an amino acid sequence of SEQ ID NO 56;
a. a' is independently a linker peptide consisting of 0-5 amino acid residues, e.g. 0-3 amino acid residues.
2. The fusion protein of claim 1, wherein the amino acids constituting the connecting peptide are selected from the group consisting of glycine, alanine, isoleucine, and valine; glycine is preferred.
3. The fusion protein of claim 1 or 2, wherein one of a and a' is N-terminally attached to the linker peptide and the other is C-terminally attached to the linker peptide.
4. The fusion protein of any one of claims 1 to 3, wherein the fusion protein has the structure:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 1-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptides2-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 4-polypeptide 2-COOH;
NH2-polypeptide 1- (GLY)5-polypeptide 4- (GLY)5-polypeptide 1-COOH;
NH2-polypeptide 1- (ALA)2-polypeptide 4- (ILE)3-polypeptide 1-COOH; or
NH2-polypeptide 1-VAL-polypeptide 4- (VAL)4-polypeptide 1-COOH;
preferably, the structure of the fusion protein is as follows:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 4-polypeptide 3-polypeptide 1-COOH;
NH2-polypeptide 1-polypeptide 3-polypeptide 1-COOH;
NH2-polypeptide 1-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 1-COOH;
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-GLY-polypeptide 4-GLY-polypeptide 2-COOH;
NH2-polypeptide 2-polypeptide 3-polypeptide 2-COOH; or
NH2-polypeptide 2-polypeptide 4-polypeptide 2-COOH;
more preferably, the structure of the fusion protein is as follows:
NH2-polypeptide 1-GLY-polypeptide 3-GLY-polypeptide 1-COOH; or
NH2-polypeptide 2-GLY-polypeptide 3-GLY-polypeptide 2-COOH.
5. A nucleic acid sequence encoding the fusion protein of any one of claims 1 to 4.
6. A vector comprising the nucleic acid sequence of claim 5.
7. A host cell comprising the vector of claim 6.
8. A method of making a fusion protein according to any one of claims 1 to 4, comprising the step of transcribing and translating the nucleic acid sequence of claim 5 under conditions in which a detectable amount of the fusion protein is expressed.
9. A method of preparing a fusion protein according to any one of claims 1 to 4, comprising the steps of:
1) constructing a nucleic acid sequence of the fusion protein according to any one of claims 1 to 4;
2) constructing an expression vector comprising the nucleic acid sequence constructed in step 1);
3) using the expression vector constructed in step 2) to transfect or transform a host cell and allowing the nucleic acid sequence to be expressed in the host cell;
4) purifying to obtain the fusion protein.
10. Use of the fusion protein according to any one of claims 1 to 4, the nucleic acid sequence of claim 5, the vector of claim 6 or the host cell of claim 7 for the preparation of a medicament for the treatment of diabetes, obesity, and/or diabetes, obesity related diseases.
11. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 4 and one or more pharmaceutically acceptable excipients.
12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is a liquid injection or a lyophilized injection.
CN2010102205671A 2010-07-08 2010-07-08 Fusion protein comprising GLP-1 (Glucagon-Like Peptide) or analog thereof and preparation method as well as application thereof Pending CN102311501A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2694095A1 (en) * 2011-04-05 2014-02-12 Longevity Biotech, Inc. Compositions comprising glucagon analogs and methods of making and using the same
CN103705446A (en) * 2012-10-08 2014-04-09 正大天晴药业集团股份有限公司 Polyene phosphatidyl choline injection, and preparation method thereof
WO2018032929A1 (en) * 2016-08-16 2018-02-22 中国药科大学 Highly active, long-acting anti-diabetic fusion protein, and manufacturing method and pharmaceutical application thereof
CN110251662A (en) * 2018-12-12 2019-09-20 四川利通科创生物医药科技有限公司 A kind of drug with antiobesity action
CN110251661A (en) * 2018-12-12 2019-09-20 四川利通科创生物医药科技有限公司 It is a kind of for treating the pharmaceutical preparation of diabetes or weight-reducing
CN117327200A (en) * 2023-11-28 2024-01-02 西宝生物科技（上海）股份有限公司 Dual-functional recombinant protein GIK for regulating and controlling glycolipid metabolism and resisting aging and preparation method thereof
WO2024141054A1 (en) * 2022-12-30 2024-07-04 苏州康宁杰瑞生物科技有限公司 Pharmaceutical composition comprising fusion protein and use thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (en) * 2000-12-07 2004-03-17 GLP-1 fusion protein
CN1935846A (en) * 2005-09-14 2007-03-28 王庆华 Fusion protein for treating diabetes, and its preparing method and use
CN101273134A (en) * 2005-07-27 2008-09-24 王庆华 Glp/1/exendin 4 igg fc fusion constructs for treatment of diabetes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (en) * 2000-12-07 2004-03-17 GLP-1 fusion protein
CN101273134A (en) * 2005-07-27 2008-09-24 王庆华 Glp/1/exendin 4 igg fc fusion constructs for treatment of diabetes
CN1935846A (en) * 2005-09-14 2007-03-28 王庆华 Fusion protein for treating diabetes, and its preparing method and use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JESSICA E. MATTHEWS ET AL: "Pharmacodynamics, Pharmacokinetics, Safety, and Tolerability of Albiglutide, a Long-Acting Glucagon-Like Peptide-1 Mimetic, in Patients with Type 2 Diabetes", 《J CLIN ENDOCRINOL METAB》 *

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* Cited by examiner, † Cited by third party
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EP2694095A1 (en) * 2011-04-05 2014-02-12 Longevity Biotech, Inc. Compositions comprising glucagon analogs and methods of making and using the same
EP2694095A4 (en) * 2011-04-05 2014-12-24 Longevity Biotech Inc Compositions comprising glucagon analogs and methods of making and using the same
US9790262B2 (en) 2011-04-05 2017-10-17 Longevity Biotech, Inc. Compositions comprising glucagon analogs and methods of making and using the same
CN103705446A (en) * 2012-10-08 2014-04-09 正大天晴药业集团股份有限公司 Polyene phosphatidyl choline injection, and preparation method thereof
WO2018032929A1 (en) * 2016-08-16 2018-02-22 中国药科大学 Highly active, long-acting anti-diabetic fusion protein, and manufacturing method and pharmaceutical application thereof
US11078250B2 (en) 2016-08-16 2021-08-03 China Pharmaceutical University High-activity long-acting hypoglycemic fusion protein as well as preparation method and medical application thereof
CN110251661A (en) * 2018-12-12 2019-09-20 四川利通科创生物医药科技有限公司 It is a kind of for treating the pharmaceutical preparation of diabetes or weight-reducing
CN110251662A (en) * 2018-12-12 2019-09-20 四川利通科创生物医药科技有限公司 A kind of drug with antiobesity action
CN110251661B (en) * 2018-12-12 2022-09-16 福州市台江区希吉亚健康科技有限公司 A medicinal preparation for treating diabetes or reducing weight
CN110251662B (en) * 2018-12-12 2023-01-31 福州市台江区希吉亚健康科技有限公司 A medicine with weight reducing effect
WO2024141054A1 (en) * 2022-12-30 2024-07-04 苏州康宁杰瑞生物科技有限公司 Pharmaceutical composition comprising fusion protein and use thereof
CN117327200A (en) * 2023-11-28 2024-01-02 西宝生物科技（上海）股份有限公司 Dual-functional recombinant protein GIK for regulating and controlling glycolipid metabolism and resisting aging and preparation method thereof
CN117327200B (en) * 2023-11-28 2024-02-09 西宝生物科技（上海）股份有限公司 Dual-functional recombinant protein GIK for regulating and controlling glycolipid metabolism and resisting aging and preparation method thereof

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