CN113214410A - Recombinant plasmid for expressing corticotropin and insulin-like growth factor 1 fusion protein and construction method of recombinant bacterium - Google Patents

Recombinant plasmid for expressing corticotropin and insulin-like growth factor 1 fusion protein and construction method of recombinant bacterium Download PDF

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CN113214410A
CN113214410A CN202110624088.4A CN202110624088A CN113214410A CN 113214410 A CN113214410 A CN 113214410A CN 202110624088 A CN202110624088 A CN 202110624088A CN 113214410 A CN113214410 A CN 113214410A
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acth
igf
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王大勇
黄永林
裴业春
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Hainan University
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    • C07K14/695—Corticotropin [ACTH]
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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Abstract

The invention relates to a construction method of recombinant plasmid and recombinant bacterium for expressing corticotropin and insulin-like growth factor 1 fusion protein. The invention adopts an optimized codon suitable for expression in engineering bacteria to construct an expression plasmid for encoding ACTH and IGF-1 fusion protein: HisTag-linker-EKst-ACTHIGF-1pET30a plasmid. After induction expression in Escherichia coli expression strain BL21-DE3, HisTag-linker-EKst-ACTH-IGF-1 polypeptide chain is separated and purified from the disrupted and dissolved inclusion body by using a nickel column, then HisTag-linker-EKst fragment is cut by using enterokinase with histidine tag, and then enterokinase and HisTag-linker-EKst fragment are removed by using a Ni column to obtain ACTH-IGF-1 fusion protein.

Description

Recombinant plasmid for expressing corticotropin and insulin-like growth factor 1 fusion protein and construction method of recombinant bacterium
The application is a divisional application with application date of 2017, 11/04, application number of 201711074686.9 and invention name of corticotropin and insulin-like growth factor 1 fusion protein and a preparation method thereof.
Technical Field
The invention belongs to the field of biological pharmacy. In particular to the primary structure of DNA and protein, the construction and expression of an expression vector, and the separation and purification of protein.
Background
Corticotropin (Corticotropin), also known as adrenocorticotropic hormone (ACTH), is a 39-amino acid polypeptide hormone secreted by the tissues of anterior lobe of pituitary, hypothalamus, adrenal medulla, intestinal tract and placenta, and has the effects of regulating biosynthesis and metabolism of sugar, fat and protein, regulating cardiovascular and cerebrovascular functions, improving resistance, regulating synthesis and secretion of Glucocorticoid (GC), recovering and regenerating nerve injury, resisting inflammation, immunosuppression, antitoxin and antishock. ACTH is the only specific medicine for clinically treating infantile convulsion under two years old, the disease incidence rate is 0.5% -1%, 6-33% of infants suffering from infantile convulsion die before 3 years old, 70-90% of infants suffer from intelligence development slowly, and 30% of infants develop autism. ACTH is also used in the treatment of lupus erythematosus, acute exacerbations of multiple sclerosis, nephrotic syndrome, systemic dermatomyositis, and sarcoidosis; also can be used for adjuvant treatment of psoriatic arthritis, rheumatic arthritis, ankylosing spondylitis, severe ocular allergy and inflammation. ACTH is unstable with an in vivo half-life of only 15 minutes; ACTH, currently used clinically, is extracted from animal tissues, is costly to produce, and has the potential to transmit animal viruses and mycoplasma. The invention prepares the gene recombinant ACTH fusion protein by expressing in engineering bacteria, has low production cost and basically cannot transmit animal viruses and mycoplasma.
Insulin-like growth factor 1 (IGF-1), also known as Somatomedin or growth hormone mediator (Somatomedin), contains a 70 amino acid polypeptide hormone with a relative molecular weight of 7649, with a half-life of up to 20 hours, and acts like insulin. The main functions of IGF-1 in human body are regulating blood sugar, promoting growth, promoting cell differentiation, wound repair, etc. The IGF-1 preparation is clinically used for treating various chronic diseases such as diabetes, insulin resistance syndrome, dwarfism, nervous system diseases and the like, and has good effect. The invention expresses and purifies ACTH and IGF-1 by fusion. The obtained fusion protein not only can enhance the action of ACTH, but also has double pharmacological actions of ACTH and IGF-1.
The initial expression architecture was HisTag-linker-EKst-ACTH-IGF-1. Wherein HisTag is a histidine tag, linker is a random connection sequence, and EKst is an Enterokinase (Enterokinase) substrate sequence (the amino acid sequence is DDDDK). The enterokinase can specifically cut the tail end of the substrate sequence amino acid sequence to obtain the ACTH-IGF-1 fusion protein without redundant amino acids.
Disclosure of Invention
1. Objects of the invention
A gene recombinant fusion protein (ACTH-IGF-1) is expressed and purified by engineering bacteria, and its preparation and purification method.
2. Technical scheme of the invention
Firstly, ACTH is connected with IGF-1 by using an overlapping PCR technology, and a cDNA framework with endonuclease sites (BamH1 and Sal1) at two ends is constructed: BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1, in which base sequence coding for enterokinase substrate (EKst) is inserted before ACTH, base sequence coding for histidine tag (HisTag) is inserted before EKst, and ACTH and IGF-1 are connected by flexible connecting sequence GGGS.
Two, double digestion BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1 and vector pET30a, resulting in sticky ends.
Thirdly, BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1 from which cohesive ends were excised was ligated to plasmid pET30a using a T4 ligase construct pET30a plasmid expressing HisTag-linker-EKst-ACTH-IGF-1 (FIG. 1).
And fourthly, transferring the constructed plasmid into an escherichia coli expression strain BL21-DE3, screening a high expression strain, and performing induced expression.
And fifthly, crushing the thalli, collecting and washing the inclusion bodies, and crushing and dissolving the inclusion bodies to obtain a solution rich in HisTag-linker-EKst-ACTH-IGF-1.
Sixthly, purifying by using a nickel (Ni) column to obtain HisTag-linker-EKst-ACTH-IGF-1.
And seventhly, cutting HisTag-EKst-ACTH-IGF-1 by using enterokinase with a His tag to obtain ACTH-IGF-1 fusion protein.
Eighthly, removing the enterokinase with the His label and the cut HisTag-linker-EKst by using a nickel column to obtain the purified ACTH-IGF-1.
3. Advantageous effects of the invention
The invention can obtain the fusion protein IGF-1-ACTH with double functions of ACTH and IGF-1, and can enhance the action of ACTH.
Drawings
FIG. 1 HisTag-linker-EKst-ACTH-IGF-1pET30a plasmid map.
FIG. 2 shows the results of the construction of the expression plasmid HisTag-EKst-linker-ACTH-IGF-1-pET30 a. a. PCR amplification of fragment BamH 1-HisTag-linker-EKst-ACTH. M: marker; lane 1: empty lane, lane 2: PCR amplification product of BamH 1-HisTag-linker-EKst-ACTH. b. PCR amplification of fragment IGF-1-Sal 1. M: marker; lane 1: empty lane, lane 2: PCR amplification product of IGF-1-Sal 1. c. PCR amplification of fragment BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal 1. M: marker; lane 1: empty lane, lane 2: PCR amplification product of BamH1-HisTaglinker- -EKst-site-ACTH-IGF-1-Sal 1. d. Cleavage of vector pET30 a. M: marker; lane 1: pET30a vector, lane 2: BamH1, Sal1 double enzyme digestion vector pET30 a. e. Enzyme digestion identification of the corticotropin-insulin-like growth factor 1 fusion protein expression vector HisTag-linker-EKst-ACTH-IGF-1-pET30 a. M: marker; lane 1: pET30a empty vector, lane 2, 3: BamH1, Sal1 double digested inserted HisTag-EKst-linker-ACTH-IGF-1-pET30a sequence plasmid.
FIG. 3 is a graph of HisTag-linker-EKst-ACTH-IGF-1 sequencing.
FIG. 4 is a graph showing the results of purification of the recombinant fusion protein HisTag-linker-EKst-ACTH-IGF-1 by a nickel column after its expression. HisTag-linker-EKst-ACTH-IGF-1-pET30a plasmid is expressed in BL21-DE3 engineering bacteria, then is subjected to ultrasonication, the supernatant is purified by a nickel column, and is separated by SDS-PAGE (15% Tris-Glycine polyacrylamide gel), and then is subjected to Coomassie brilliant blue R-250 in-situ staining. M: and (4) marking molecular weight. Lanes 1-14: and the components 4 to 13 are the fusion protein HisTag-linker-EKst-ACTH-IGF-1 obtained by Ni column purification.
FIG. 5 Western blot assay of purified ACTH-IGF-1. ACTH-IGF-1 was separated by SDS-PAGE (10% Tris-tricine gel) and transferred to a PVDF membrane with low fluorescence background for detection, with the primary antibody being a mouse anti-human ACTH (Santa Cruz) -specific antibody and the secondary antibody being Alexa488 fluorescently labeled goat anti-mouse antibody (Abcam), and the fluorescence band being detected with typhoon FLA 9500(GE healthcare).
Detailed Description
The first embodiment is as follows: the embodiment expresses, separates and purifies the gene recombinant ACTH-IGF-1 fusion protein, and comprises the following steps:
firstly, an ACTH CDs sequence is optimally designed according to codon preference in a prokaryotic expression system so as to be suitable for high-efficiency expression in an escherichia coli expression strain BL21-DE 3. The method comprises the steps of taking BamH1 and Sal1 as enzyme cutting sites at the upstream and downstream ends of a CDs sequence of a fusion expression protein, constructing a prokaryotic expression sequence HisTag-linker-EKst-ACTH-IGF-1-Sal1 of the fusion protein with a His tag and an enterokinase enzyme cutting site, wherein the nucleotide sequence is shown as SEQ ID No.1, and the amino acid sequence of the fusion protein with the His tag and the enterokinase enzyme cutting site is shown as SEQ ID No. 2. The method comprises the following specific steps:
designing a primer: designing an upstream primer BamH1-HisTag-linker-EKst-ACTH-F and a downstream primer ACTH-R for amplifying BamH 1-HisTag-linker-EKst-ACTH; an upper primer IGF-1-F and a lower primer IGF-1-Sal1-R for IGF-1 amplification are designed. The primer sequence is shown in a nucleotide sequence table.
Amplifying BamH1-HisTag-linker-EKst-ACTH (shown in figure 2a) by using primers BamH1-HisTag-linker-EKst-ACTH-F (shown in SEQ ID NO. 3) and ACTH-R (shown in SEQ ID NO. 5) and artificially synthesized ACTH sequence as a template by using an overlap PCR method; using a plasmid containing IGF-1 gene as a template, and amplifying IGF-1-Sal1 (shown in a figure 2b) by using a primer IGF-1-F (shown in a SEQ ID NO. 4) and a primer IGF-1-Sal1-R (shown in a SEQ ID NO. 6); finally, using the two amplified fragments (BamH1-HisTag-EKst-ACTH and IGF-1-Sal1) as templates, and using primers BamH1-HisTag-EKst-ACTH-F (nucleotide sequence shown in SEQ ID NO. 3) and IGF-1-Sal1-R (nucleotide sequence shown in SEQ ID NO. 6) to amplify the target fragment: BamH1-HisTag-EKst-ACTH-IGF-1-Sal1 (see FIG. 2c), the nucleotide sequence between ACTH and IGF-1 encoding the amino acid flexible linker GGGS.
Secondly, the fragment BamH1-HisTag-EKst-ACTH-IGF-1-Sal1 and the blank vector pET30a were digested with BamH1 and Sal1, respectively (see FIG. 2 d).
Thirdly, the fragments and the vector are connected by T4DNA ligase to construct a plasmid HisTag-Linker-EKst-ACTH-IGF-1-pET30 a. Enzyme digestion identification (see fig. 2e), drawing a fusion protein expression plasmid map (see fig. 1), and sequencing results are shown in fig. 3.
And fourthly, transferring the plasmid HisTag-linker-EKst-ACTH-IGF-1-pET30a into an escherichia coli expression strain BL21-DE3 at 40 ℃ for 30 seconds, recovering the thallus at 37 ℃ for 1 hour, coating 50uL of the recovered thallus on an LB solid culture plate containing kanamycin, and carrying out inverted culture at 37 ℃ for 16 hours. A single colony on the plate was picked and transferred to a conical flask containing 50mL of liquid LB medium containing kanamycin and shaken at 37 ℃ until OD 600 became 0.6, and expressed at 180rpm and 37 ℃ for 5 hours by adding 0.05mM IPTG.
Fifthly, collecting the engineering bacteria in a centrifuge tube, centrifuging for 15min at the temperature of 4 ℃ at the rpm of 12,000, and discarding the supernatant. The cells were gently washed with non-denaturing lysis solution (50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0), and the pellet was collected by centrifugation at 12,000rpm at 4 ℃ for 10min and washed 3 times for 15min each. Adding 8mL of non-denatured lysate per gram of wet weight, and dispersing the thalli by using a rotary mixer. And (3) putting the bacterial liquid on ice, crushing the thalli by using an ultrasonic crusher, wherein the crushing power is 400W, and crushing is stopped for 3s and 5s for 200 times. Centrifuge at 12,000rpm at 4 ℃ for 20 min. The pellet was suspended in 9-fold volume of inclusion body wash I (50mM Tris-HCl, 100mM NaCl, 2mM EDTA, 1mM DTT, 0.5% (v/v) Triton X-100, pH 8.0) under ice-bath conditions, sonicated for 3X 10 seconds, allowed to stand at room temperature for 10min, centrifuged at 4 ℃ at 12,000rpm for 20min, and the supernatant and pellet were collected, respectively. The pellet was suspended in 9-fold volume of inclusion body wash II (50mM Tris-HCl, 100mM NaCl, 2mM EDTA, 1mM DTT, pH 8.0), mixed well, left at room temperature for 10min, centrifuged again for 20min under the same conditions, and the combined supernatants were collected for protein purification.
Sixthly, the regenerated NI column is washed by 2 times of 20 percent ethanol by volume and is washed by 2V of deionized water. The mobile phase was exchanged for equilibration buffer (50mM Tris-cl, 300mM NaCl, 10mM imidazole, pH 8.0) and equilibrated for 5 column volumes. The mobile phase was changed to a rinse (50mM Tris-cl, 300mM NaCl, 100mM imidazole, pH 8.0) and 40 column volumes were rinsed. Elution was performed by replacing the mobile phase with the eluent (50mM Tris-cl, 300mM NaCl, 250mM imidazole, pH 8.0), and fractions of the eluent were collected and subjected to R-250 staining after electrophoresis on 15% SDS-PAGE (see FIG. 4).
Seventhly, adding the enterokinase with His labels into the eluate respectively, wherein the final concentration is 1mM, and incubating the mixture for 1 hour at 37 ℃ to cut HisTag-linker-EKst-ACTH-IGF-1 polypeptide chains in the sample to obtain free ACTH-IGF-1 and His-Tag-linker-EKst.
And eighthly, balancing the Ni column by adopting the method in the sixth step, adding the eluent components after the enterokinase enzyme digestion into the Ni column, and standing for 30-60min at 4 ℃ to remove the enterokinase connected with the His label and the HisTag-linker-EKst polypeptide in the enzyme digestion eluent. The result of identifying ACTH-IGF-1 in each eluate component by Western blot is shown in FIG. 5.
The second embodiment is as follows: the present embodiment differs from the present embodiment in that the pET30a plasmid is not used, but pET21a is used as an expression vector.
The third concrete implementation mode: this embodiment differs from the first or second embodiment in that rather than using the pET30a or pET21a plasmid, pET28a is used as the expression vector.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (3)

1. A construction method of a recombinant plasmid for expressing a fusion protein of human corticotropin and insulin-like growth factor 1 is characterized in that a nucleotide sequence of the recombinant plasmid contains a nucleotide sequence for coding the human corticotropin and the insulin-like growth factor 1, and the construction method comprises the following steps:
1) ACTH was ligated to IGF-1 using overlap PCR to construct a cDNA construct with endonuclease sites at both ends: BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal 1; the enzyme cutting sites are BamH1 and Sal 1; a base sequence coding an enterokinase substrate EKst is inserted in front of ACTH in the cDNA framework, a base sequence coding a histidine tag HisTag is inserted in front of EKst, and the ACTH is connected with IGF-1 through a flexible connecting sequence GGGS.
2) Double digestion BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1 and vector pET30a, resulting in sticky ends;
3) the cohesive end-cleaved BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1 and plasmid pET30a were ligated by using a T4 ligase construct to construct a recombinant plasmid expressing HisTag-linker-EKst-ACTH-IGF-1.
2. A construction method of a recombinant bacterium for expressing a corticotropin and insulin-like growth factor 1 fusion protein comprises the following steps:
transferring the recombinant plasmid constructed in the claim 1 into an escherichia coli expression strain BL21-DE3, and screening high-expression recombinant bacteria.
3. A method for preparing a corticotropin and insulin-like growth factor 1 fusion protein based on the recombinant bacterium of claim 2, comprising the steps of:
s1, inducing the recombinant bacteria to express;
s2, crushing thalli, collecting and washing an inclusion body, and crushing and dissolving the inclusion body to obtain a solution rich in HisTag-linker-EKst-ACTH-IGF-1;
s3, purifying HisTag-linker-EKst-ACTH-IGF-1 in the solution by using a nickel (Ni) column;
s4, cutting the HisTag-EKst-ACTH-IGF-1 by using enterokinase with a His label to obtain ACTH-IGF-1 fusion protein;
s5, removing the enterokinase with the His label and the cut HisTag-linker-EKst by using a nickel column to obtain the purified ACTH-IGF-1.
CN202110624088.4A 2017-11-04 2017-11-04 Recombinant plasmid for expressing corticotropin and insulin-like growth factor 1 fusion protein and construction method of recombinant bacterium Pending CN113214410A (en)

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