CN114644715B - Preparation method of fusion protein and complex of IGFBP-3 and IGF-1 - Google Patents
Preparation method of fusion protein and complex of IGFBP-3 and IGF-1 Download PDFInfo
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- CN114644715B CN114644715B CN202210213262.0A CN202210213262A CN114644715B CN 114644715 B CN114644715 B CN 114644715B CN 202210213262 A CN202210213262 A CN 202210213262A CN 114644715 B CN114644715 B CN 114644715B
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
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- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
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Abstract
The application discloses a fusion protein of IGFBP-3 and IGF-1 and a preparation method of a complex, wherein the fusion protein is IGFBP-3-Glu-IGF-1, glutamic acid forms a specific dipeptide bond with IGFBP-3 and IGF-1 through hydroxylamine cleavage, and IGFBP-3 and IGF-1 can be released under the condition of hydroxylamine solution, so that the preparation of the complex of IGFBP-3 and IGF-1 is realized. The preparation process of the complex of IGFBP-3 and IGF-1 fusion protein can be simplified, and the method for maintaining the activity of the complete IGFBP-3 and IGF-1 has wide application prospect.
Description
Technical Field
The application belongs to the technical field of biological medicines, and relates to a preparation method of a fusion protein of IGFBP-3 and IGF-1 and a compound.
Background
Insulin-like growth factor-1 (IGF-1) is an active protein polypeptide substance, and is extracted from human serum in 1976 at RINDERKNECTH, and is named after the fact that the structure of the insulin-like growth factor-1 is similar to insulin, and research on IGF-1 is widely conducted by scientific research institutions at home and abroad since 1976. The current market is approved by IGF-1 as a drug, and only the American 2005 approved commercial mecamylamine-Lin Feipei is a binary protein complex composed of recombinant human insulin-like growth factor-1 (rh-IGF-1) and recombinant human insulin-like growth factor binding protein-3 (rh-IGFBP-3) and is used for treating patients with serious primary IGF-1 or growth hormone deficiency.
Numerous research data indicate that IGF-1 plays an important therapeutic role in the treatment of diabetes, muscular dystrophy, arthritis, osteoporosis, amyotrophic Lateral Sclerosis (ALS) and other diseases, IGFBP-3 is the main IGF-1 carrier in blood, and in vitro experimental research shows that IGFBP-3 can promote or inhibit the action of IGF-1. IGFBP-3 has a higher affinity for IGF-1 than for IGF-1 receptor and IGF-1, and has an important regulatory effect on IGF-1 biological activity, so that complexes of insulin growth factor-1 (IGF-1) and insulin growth factor binding protein-3 (IGFBP-3) are widely used in the medical field.
The technology for preparing IGF-1 biological medicine by a genetic engineering method is quite mature, has great advantages compared with direct extraction in an animal body, can realize continuous mass production and reduce the production cost. Meanwhile, the genetically engineered medicine can also prolong the half life of the medicine, can be prepared into a long-acting medicine preparation, avoids frequent administration, greatly reduces the pain of patients, and can overcome the defect of poor compliance of the patients with frequent administration, thereby being more easily accepted. The pET series vector in genetic engineering is an expression system which is widely applied in the current prokaryotic expression. Although the pET-32a expression product contains specific cleavage sites of enterokinase and thrombin, the enzyme reagent is expensive and is unfavorable for industrial production, and the upstream of the target protein after cleavage has tens to tens of additional amino acid sequences, so that the renaturation effect on the small molecular weight protein is great and the activity of the protein is directly influenced. Therefore, the existing production of insulin growth factor-1 (IGF-1) and insulin growth factor binding protein-3 (IGFBP-3) complex is always a difficult problem to be solved by manufacturers, and the production process of the complex needs to be advanced to realize pipeline processing, so that the preparation process is particularly important.
Content of the application
The present application aims at providing a method for producing complex of insulin growth factor-1 (IGF-1) and insulin growth factor binding protein-3 (IGFBP-3) with relatively simple production process and maintained activity of complete IGFBP-3 and IGF-1.
In order to achieve the technical purpose and effect, the application is realized by the following technical scheme:
as one embodiment of the present application, there is provided a fusion protein comprising IGFBP-3 and IGF-1, said fusion protein comprising IGFBP-3 and IGF-1 and glutamic acid (Glu) located at a dipeptide bond forming a linkage between IGFBP-3 and IGF-1.
In embodiments of the application IGFBP-3 and IGF-1 are linked by cleavable dipeptide linkages to form a fusion protein that is more stable and expressed in an active and soluble manner than IGFBP-3 and IGF-1 alone. Simultaneously cleavable dipeptide bonds can be cleaved in the lysate to release IGFBP-3 and IGF-1, preparing a complex solution comprising IGFBP-3 and IGF-1.
The dipeptide bond is a specific cleavage peptide bond of hydroxylamine.
The IGFBP-3, glutamic acid and IGF-1 are sequentially connected to form the fusion protein, namely the N end of the fusion protein is IGFBP-3, and the C end of the fusion protein is IGF-1.
The amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
In an embodiment of the application the terminal amino acid of IGFBP-3 is lysine (Lys), the lysine is a basic amino acid, the side chain structure is H 2N-(CH2)4 -, the glutamic acid is an acidic amino acid, the side chain structure is HOOC- (CH 2)2 -, the side chain structure H 2N-(CH2)4 -and HOOC- (CH 2)2 -can form an amide bond (-CO-NH 2) through condensation, meanwhile, the first amino acid of IGF-1 is glycine (Gly), glycine (Gly) is aliphatic, the side chain structure is H-, and the side chain structure can form a specific cleavage peptide bond of hydroxylamine with the amide bond (-CO-NH 2).
As an embodiment of the present application, there is also provided a method for constructing the fusion protein, comprising:
inserting the fusion protein gene into an expression vector to obtain an expression plasmid;
engineering bacteria obtained by transforming the expression plasmid by using competent cells;
inducing the engineering bacteria to express and crack the obtained fusion protein.
In one embodiment of the present application, IGFBP-3 and IGF-1 gene are expressed in tandem by genetic engineering and a self-cleavable linker peptide is constructed between IGFBP-3 and IGF-1, followed by fermentation and purification by E.coli to obtain IGFBP-3 and IGF-1 fusion protein. Since the first amino acid of IGFBP-3 and IGF-1 is glycine, the application introduces glutamic acid, glycine and lysine dipeptide bond, the formed dipeptide bond has hydroxylamine cleavage function, after IGFBP-3 and IGF-1 are expressed in series, the complex of IGFBP-3 and IGF-1 is obtained through cleavage, the preparation of two proteins can be realized by one-step purification, the properties of IGFBP-3 and IGF-1 are not influenced, and the activities of the complete IGFBP-3 and IGF-1 are maintained.
Further, the expression vector is a pET expression system, preferably a vector pET32 alpha (+).
Further, the competent cells are E.coli competent cells, preferably E.coli DH 5. Alpha. Competent cells.
Further, the method further comprises the step of purifying the fusion protein obtained by cleavage, wherein the purification method is selected from salting out and organic solvent precipitation, electrophoresis, dialysis, chromatography, molecular sieve or ultracentrifugation.
As one embodiment of the present application, there is also provided a method for preparing IGFBP-3 and IGF-1 complex, wherein the fusion protein is cleaved in hydroxylamine solution.
The cleavage conditions of the fusion protein are: hydroxylamine concentration was 3m, ph was 9.0, reaction temperature was 40 ℃, and reaction time was 8h.
In an embodiment of the present application, the IGFBP-3 and IGF-1 dipeptide bond is linked by cleavage of the hydroxylamine solution, thereby releasing IGFBP-3 and IGF-1, and the complex solution containing IGFBP-3 and IGF-1 is obtained directly, and the preparation of the IGFBP-3 and IGF-1 complex is accomplished by concentration and drying.
As another embodiment of the present application, there is also provided an engineering bacterium comprising the gene of the fusion protein.
Designing a primer for amplification to obtain the gene of the fusion protein, inserting the gene of the fusion protein into an expression vector, and transforming by using escherichia coli.
As another embodiment of the present application, there is also provided an expression plasmid comprising the gene of the fusion protein.
Preferably, the vector of the expression plasmid is a pET expression system, preferably a vector pET32 alpha (+).
The beneficial effects of the application are as follows:
The application has great application value, and the fusion protein of IGFBP-3 and IGF-1 is obtained by adding a specific cleavage site between IGFBP-3 and IGF-1 and purifying for one time, and the fusion protein is cleaved to release IGFBP-3 and IGF-1 monomers to prepare a compound solution. The method not only can obtain a relatively stable product, but also has simple and low-cost separation method, reduces the production cost and has wide application prospect.
Drawings
FIG. 1 is an electrophoretogram of the fusion protein IGFBP-3-Glu-IGF-1 of the application.
Detailed Description
The following description of the present application will be made more complete and clear in view of the detailed description of the application, which is to be taken in conjunction with the accompanying drawings that illustrate only some, but not all, of the embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The main technical content of the application is to construct a fusion protein expression vector through the design and synthesis of recombinant human IGFBP-3 and IGF-1 fusion protein related genes, prepare engineering bacteria and prepare IGFBP-3 and IGF-1 fusion proteins through a fermentation method. The two fusion proteins are IGFBP-3 and IGF-1, the two proteins are connected through a dipeptide bond, the fusion proteins have better stability than the single IGFBP-3 and IGF-1, and are expressed in an active and soluble mode; dipeptide bond as specific cleavage site can be cleaved under physiological conditions to release IGFBP-3 and IGF-1, and a complex solution containing IGFBP-3 and IGF-1 is prepared.
IGFBP-3 and IGF-1 of the application may be natural or wild-type proteins and mutants and derivatives thereof, preferably IGF-1 in the application is derived from mature native human insulin growth factor-1 (IGF-1) having an amino acid sequence as shown in SEQ ID NO. 2; IGFBP-3 is derived from mature native insulin growth factor binding protein-3 (IGFBP-3) and has the amino acid sequence shown in SEQ ID NO. 3.
The mutant of FGF21 and IGF-1 refers to mature FGF21 protein or IGF-1 protein with at least one amino acid mutation. In general, mutants possess a modified function, structure or property relative to the wild-type protein. For example: the mutant has better stability in solution, is not easy to form aggregation and precipitation, and maintains the biological activity; or the mutant body can be better compatible with the components of the pharmaceutical preparation, so that various pharmaceutical products can be prepared later, and the products can maintain the physiological and chemical characteristics of the medicine and the biological activity of the product during storage; alternatively, the degree of glycosylation of the mutant is reduced, and batch-to-batch consistency is ensured while maintaining its functional activity. The mutation included in the mutant of the present application is not limited to the above-mentioned various definitions, and the mutant of the present application refers to a change in more than one aspect of its property, function and the like relative to the wild-type protein.
The dipeptide bond as a linking peptide in the present application needs to satisfy the following two conditions: 1) The polypeptide chains of the individual functional proteins should not interact with each other, nor should they fold or entangle with each other, resulting in loss of activity from each other; 2) The active centers of the functional proteins are far away from each other, so that steric hindrance is not formed to influence the activity. In addition, the requirements of protein expression, purification, renaturation efficiency and the like should be fully considered when designing the connecting peptide. It is generally believed that the introduction of a linker peptide allows for successful expression of the fusion protein, since the linker peptide can properly separate the two domains and thus avoid interference of the different domains during the reaction.
The connecting peptide can be classified into flexible connecting peptide, rigid connecting peptide and cleavable connecting peptide, and the dipeptide bond as connecting peptide in the present application belongs to cleavable connecting peptide, and the dipeptide bond is based on IGFBP-3 end and first amino acid of IGF-1, and can be used for realizing the connection of IGFBP-3 and IGF-1 on one hand and realizing the cleavage separation of IGFBP-3 and IGF-1 fusion protein on the other hand by forming a cleavable dipeptide bond with another amino acid, and realizing the preparation of IGFBP-3 and IGF-1 complex under physiological conditions.
The other amino acid refers to a generalized amino acid, and comprises natural or non-natural amino acids, mutants and derivatives. Generalized amino acids include, in particular, such as naturally occurring L-amino acids and D-amino acids, chemically modified amino acids such as mutants and derivatives; naturally occurring amino acids such as nonprotogenic amino acids, e.g., norleucine, beta-alanine, ornithine, and the like; chemically synthesized compounds having amino acid properties. Non-naturally occurring amino acids such as: alpha-methylated amino acid, D-amino acid, histidine-like amino acid, amino acid having a side chain containing an excess methylene group, amino acid having a side chain carboxyl group substituted with a sulfonic acid group, and the like.
The other amino acid forming a dipeptide bond with glycine in the present application is preferably a naturally occurring amino acid, preferably glutamic acid.
In the present application, IGFBP-3 and IGF-1 genes are fused by restriction enzyme cleavage for the construction of the fusion protein. And respectively carrying out double enzyme digestion on the fusion protein gene and the expression vector, mixing and connecting the digested plasmid and the digested gene to obtain an expression plasmid, transferring the expression plasmid into competent cells for culture, collecting thalli to extract the plasmid, carrying out PCR amplification, and preserving strains with correct sequencing.
The expression vector is a vector which is formed by adding expression elements (such as a promoter, RBS, terminator and the like) on the basis of the basic skeleton of a cloning vector in genetic engineering so that a target gene can be expressed. The pET system is the most powerful system for cloning and expressing recombinant proteins in e.coli, and in pET vectors, the gene of interest is cloned under the control of strong transcription and translation signals of T7 phage and expression is induced by providing T7RAN polymerase by the host cell.
Competent cells can induce cells by physicochemical methods to take up DNA molecules in the surrounding environment so that they are in a physiological state where they optimally take up and hold foreign DNA. The main principle is that the permeability of the cells is increased by treatment, so that exogenous genes or vectors can enter competent cells conveniently, and the holes can be repaired by the cells due to the fluidity of cell membranes.
In the application, DH5 alpha competent cells are adopted as hosts of an expression vector pET32 alpha (+), wherein the DH5 alpha competent cells are prepared by adopting escherichia coli DH5 alpha strains. The pET32 alpha (+) vector is expressed by using an escherichia coli DH5 alpha T7 phage transcription system, and the T7RNA polymerase is a high-activity RNA polymerase, and the mRNA synthesis speed is about 5 times faster than that of the escherichia coli RNA polymerase, so that the mRNA is preferentially expressed in the escherichia coli in-vivo T7 phage transcription system.
Specifically, the preparation method of IGFBP-3 and IGF-1 complex of the application comprises the following steps:
1) Design of fusion proteins
A glutamic acid (Glu) is added between IGFBP-3 and IGF-1 to form a fusion protein. The terminal amino acid of IGFBP-3 is lysine (Lys), lysine is basic amino acid, the side chain structure is H 2N-(CH2)4 -, glutamic acid is acidic amino acid, the side chain structure is HOOC- (CH 2)2-,H2N-(CH2)4 -and HOOC- (CH 2)2 -can form an amide bond (-CO-NH-) through condensation), the first amino acid of IGF-1 is glycine (Gly), glycine (Gly) is aliphatic, the side chain structure is H-, and can form a specific cleavage peptide bond of hydroxylamine with the amide bond.
2) Synthesis of fusion proteins
Primers were designed based on the designed gene sequence of the fusion protein, and EcoRI and HindIII restriction enzyme sites were added to the 5 'and 3' ends of the primers, respectively.
The target gene is amplified by PCR method. And (3) carrying out 1% agarose gel electrophoresis on the PCR product after amplification, and recovering the PCR product through a gel recovery kit after electrophoresis.
The amplification procedure was: pre-denaturation at 95℃for 5min; denaturation at 95℃for 1min; renaturation is carried out at 64 ℃ for 1min; the reaction was carried out at 72℃for 1min for a total of 35 cycles with a final reaction extension time of 10min.
3) Insertion of target gene into expression vector
The recovered PCR product was digested with EcoRI and HindIII, the DNA fragment was recovered by agarose gel electrophoresis after digestion, the pET 32. Alpha. (+) plasmid was digested with EcoRI and HindIII, and the recovered DNA fragment was ligated with the digested pET 32. Alpha. (+) vector by mixing.
After the ligation reaction was completed, the reconstituted pET32 alpha (+) vector was added to the prepared competent DH5 alpha E.coli, and then inoculated onto an ampicillin-containing agarose plate for resistance selection. Single bacterial colony is selected for fermentation, bacterial body is collected for extracting plasmid, the PCR primer is adopted for amplification, the amplified product is subjected to DNA sequence determination, and the strain with correct sequencing is stored in a glycerol pipe at the temperature of minus 80 ℃.
4) Induction expression of engineering bacteria
Inoculating strain stored in-80deg.C refrigerator into LB culture medium containing amp+ with inoculum size of one fungus ring, culturing at 37deg.C and 220rpm, inoculating to fermentation culture medium with inoculum size of 5% when absorbance of bacterial suspension at OD 600 nm is about 0.8, culturing at 37deg.C and 220rpm until OD 600 nm is about 0.6-0.8, adding IPTG to final concentration of 0.5mmol/L, culturing at 37deg.C and 220rpm for 8 hr, and collecting bacterial cells.
5) Coli disruption
And (3) taking the thalli collected after the fermentation broth is centrifuged, adding the bacterial lysate to resuspend the thalli, and carrying out bacterial breaking treatment by using an ultrasonic cell disruption instrument. During the bacterial breaking, the bacterial suspension is placed in an ice bath environment, the bacterial breaking program is 500W, the working time is 4s, the gap is 6s, and the ultrasonic breaking is stopped when the bacterial suspension is changed from thick to transparent.
Centrifuging the thallus suspension after ultrasonic crushing, collecting precipitate, adding a denaturing solution at 4 ℃ overnight, slowly adding a renaturation solution after inclusion body is denatured, standing at 37 ℃ for 2h, centrifuging at 10000rpm and 4 ℃ for 10min, collecting supernatant, and filtering the supernatant with a filter membrane for subsequent purification.
6) Preparation of fusion proteins
Placing a proper amount of Ni-NTA resin (the fusion protein sampled per ml of resin is less than 15 mg) into a chromatographic column, opening an end cap under the column to naturally drain liquid, adding a phosphate buffer solution for balancing, adding a bacterial lysate, and then carrying out stage elution by using phosphate buffers with different concentration gradients of imidazole after 30min, wherein the flow rate is 2ml/min, and the eluent is not collected. Finally, eluting with buffer solution containing imidazole at a flow rate of 1m1/min, collecting eluate, freeze drying, and preserving at-20deg.C.
7) Hydroxylamine cleavage reaction
The fusion protein obtained after ion exchange is added into hydroxylamine lysate, the hydroxylamine concentration is 3M, the pH is 9.0, the reaction temperature is 40 ℃, and the reaction time is 8 hours. After the completion of the reaction, the reaction mixture was dialyzed overnight against a Tris-HCI solution at 4℃and 20mM and pH8.0, freeze-dried and stored at-20 ℃.
EXAMPLE 1 cloning of the genes comprising IGFBP-3 and IGF-1 fusion protein and construction of recombinant expression vectors therefor
1) The fusion protein IGFBP-3-Glu-IGF-1 is designed, the fusion protein IGFBP-3-Glu-IGF-1 gene is synthesized, and the whole nucleic acid sequence is subjected to total gene synthesis in the biological engineering (Shanghai) limited company.
2) Removing 60 mu LPCR product gene, adding 5 times volume of TE buffer, sucking and beating by a pipetting gun, mixing uniformly, adding equal volume of phenol, mixing uniformly, centrifuging at room temperature of 12000r/min for 5min, taking supernatant, adding 1/10 volume of 3mol/LCH 3 COOK buffer (pH 5.2) and 2.5 times volume of ice-cold absolute ethyl alcohol, standing at-20 ℃ for 30min, centrifuging at 12000r/min for 5min, discarding supernatant, adding 1ml of ice-cold 70% ethyl alcohol, centrifuging at 12000r/min for 5min, discarding supernatant, drying at room temperature, adding 30 mu LTE buffer for dissolving, and obtaining PCR purified fragments.
3) The purified fusion gene and shuttle expression vector pET32 alpha (+) were subjected to double cleavage treatment with two identical restriction enzymes (EcoRI and HindIII), and the treated fragments were recovered from the target gene fragments using agarose gel recovery kit, and finally ligated using T4DNALIGASE (purchased from NEB).
The target gene and the expression vector plasmid are all processed according to the following enzyme digestion system:
Incubate at 37℃for 1h.
The connection system of the target gene and the expression vector is as follows:
overnight ligation at 4 ℃.
4) Adding 10 mu L of the ligation product into 200 mu L of competent cells E.coli (DH 5 alpha), placing on ice for 30min, carrying out heat shock for 90s at 42 ℃, rapidly taking out ice bath for 1-2 min, adding 800 mu LLB culture medium into a centrifuge tube, carrying out shaking culture at 200rpm at 37 ℃ for 1-1.5 h, centrifuging at 7000rpm for 1min, absorbing 700 mu L of supernatant, blowing the rest transformation product uniformly by a micropipette, coating the transformation product on an agarose plate containing ampicillin, carrying out resistance screening, placing at room temperature for 3-5 min, and carrying out inversion culture at 37 ℃ until single colony is visible after liquid absorption.
Single bacterial colony is selected for fermentation, bacterial body is collected for extracting plasmid, PCR is carried out for amplification, the amplified product is determined, and the strain with correct sequencing is stored in a glycerol pipe at the temperature of minus 80 ℃. As shown in SEQ ID NO.1, the fusion gene synthesized in the embodiment is successfully embedded into an expression vector pET32 alpha (+), and a DNA sequencing result shows that the recombinant pET32 alpha (+)/IGFBP-3-Glu-IGF-1 is successfully constructed.
EXAMPLE 2 construction of engineering bacteria and expression of fusion protein IGFBP-3-Glu-IGF-1
The recombinant vector with the correct sequencing result in the example 1 is transformed into a competent cell of an expression host escherichia coli DH5 alpha, and positive clones are selected and cultured in an agarose solid medium containing ampicillin overnight to obtain the recombinant strain. The method comprises the following specific steps:
1) Taking out competent cells of Escherichia coli DH5 alpha from a refrigerator at-80 ℃, and putting the competent cells on ice for melting;
2) Taking 50 mu L of competent cells under a sterile state, and placing the competent cells into a 1.5mL centrifuge tube after sterilization;
3) 10. Mu.L of ligation product was added and left in ice for 30 minutes;
4) Standing at 42 ℃ for 30 seconds (heat shock);
5) Rapidly transferring the centrifuge tube into ice and placing the centrifuge tube into ice for 2-3 min;
6) Coating on an ampicillin-containing agarose plate culture medium, and reversely culturing for 12-16 h at 37 ℃ to obtain the recombinant strain.
The recombinant strain obtained is inoculated into LB culture medium containing amp+ with a bacterial loop, cultured at 37 ℃ and 220rpm, inoculated into fermentation culture medium with an inoculum size of 5% when the absorbance of bacterial suspension at OD 600 nm is about 0.8, cultured at 37 ℃ and 220rpm until OD 600 nm is about 0.6-0.8, and induced and cultured for 8h with IPTG with a final concentration of 0.5mmol/L at 37 ℃ and 220 rpm.
After induction, taking culture supernatant to carry out SDS-PAGE electrophoresis detection, wherein the specific process is as follows:
1) Experimental materials
Protein samples before and after IPTG induction.
2) Reagents and formulation
30% Acrylamide stock, 1.5mol/LTris-HCl (pH 8.8), 1.0 mol/LTris-HCl (pH 6.8), 1 XTris-Gly running buffer (pH 8.3), decolorization solution, staining solution, 10% SDS 10mL, 10% ammonium persulfate, TEMED, 4 Xprotein loading buffer (lo adingbuffer), protein marker (protein molecular weight standard), isopropanol, dithiothreitol (DTT), deionized water, bromophenol blue indicator.
3) Experimental instrument
Vertical plate electrophoresis tank, steady voltage and steady flow electrophoresis apparatus, constant temperature water bath, gel imaging system, and micropipette (20. Mu.L, 1000. Mu.L).
4) Experimental method
SDS-PAGE gels were prepared: firstly, washing the electrophoresis glass plate with water, then wiping with alcohol, and airing. The glass plate was mounted as described and checked for leakage on three sides with deionized water. If the liquid leaks, the glass plate is reinstalled to test the liquid again. The separation gel was prepared as shown in Table 1, and finally TEMED was added, the mixture was swiftly rotated in one direction (to prevent the generation of bubbles), poured into the gap between two glass plates (to the upper edge of the red plate) with a 1000. Mu.L micropipette, and then carefully covered with a layer of isopropyl alcohol on the gel surface to seal. Standing at room temperature for about 40min until the separation gel is polymerized, pouring out the isopropanol covering liquid, and flushing the surface of the separation gel with deionized water for 3-4 times to clean the unpolymerized polyacrylamide gel. The deionized water was poured off, and the residual moisture was sucked dry with a water-absorbent paper. The concentrated gums were prepared as shown in table 1 and finally TEMED was added and mixed rapidly with rotation. Slowly filling the gap between the glass plates, inserting a sample comb (1 cm on the side facing inwards, inserting one side first, then pressing the other side from one side to the other side to discharge bubbles), and standing vertically at room temperature. After about 30min polymerization, the comb was pulled out in parallel, the glass plate was removed, and the rubber strip sealing the glass clamping plate was torn off. The concave surface of the glass clamping plate is tightly adhered to the electrophoresis tank, and the electrophoresis tank is fixed by a clamp.
Table 1 preparation method of separating gel and concentrated gel
SDS-PAGE electrophoresis: 300mL of electrophoresis buffer was added to the electrophoresis tank, and 10. Mu.L of IPTG-induced supernatant and 5. Mu.L of protein marker were sequentially added from the loading well. The cover is covered and connected with a power supply (red is connected with red and black is connected with black), and the voltage is slowly regulated to 80V to carry out electrophoresis. When the isobromophenol blue indicator reaches the separation gel, the voltage is increased to 150V, and the electrophoresis can be stopped when the blue band of the indicator migrates to the bottom edge of the gel.
Coomassie brilliant blue staining and decolorizing: the glass plate was removed from the electrophoresis tank, the gel was removed by prying it apart, the concentrated gel was removed, the gel was placed in a petri dish, and approximately 40mL of staining solution was added and stained on a destaining shaker for 50min. After dyeing, the dyeing liquid is recovered, the glue is washed for 2 to 3 times by water, and then the decoloring liquid is added for decoloring until clear protein bands are seen.
As a result, it is clearly shown in FIG. 1 that the fusion protein IGFBP-3-Glu-IGF-1 was obtained.
EXAMPLE 3 preparation of IGFBP-3 and IGF-1 complex solution
1) 600ML of the fermentation broth of example 2 was centrifuged to collect the cells, and 30mL of the bacterial lysate was added to resuspend the cells, followed by disruption treatment using an ultrasonic cytobreaker. During the bacterial breaking, the bacterial suspension is placed in an ice bath environment, the bacterial breaking program is 500W, the working time is 4s, the gap is 6s, and the ultrasonic breaking is stopped when the bacterial suspension is changed from thick to transparent.
2) Centrifuging the thallus suspension after ultrasonic disruption, collecting precipitate, adding 5mL of denatured solution at 4 ℃ overnight, slowly adding the renaturation solution after the inclusion body is denatured, standing at 37 ℃ for 2h, centrifuging at 10000rpm and 4 ℃ for 10min, collecting supernatant, and filtering the supernatant by a 0.45 mu m filter membrane for subsequent purification.
3) Placing a proper amount of Ni-NTA resin (the fusion protein loaded on each ml of resin is less than 15 mg) into a chromatographic column, opening an end cap under the column to naturally drain liquid, adding a phosphate buffer solution for balancing, adding a bacterial lysate, and performing stage elution by using 30m1 of phosphate buffer solution respectively containing 0,10, 20, 50 and 100mM imidazole after 30min, wherein the flow rate is 2ml/min, and the eluent is not collected. Finally, eluting with buffer solution containing 400mM imidazole at a flow rate of 1ml/min, collecting eluate, placing into a concentration tube, centrifuging, concentrating, freeze drying, and preserving at-20deg.C.
4) The fusion protein obtained after ion exchange is added into hydroxylamine lysate, the hydroxylamine concentration is 3M, the pH is 9.0, the reaction temperature is 40 ℃, and the reaction time is 8 hours. After the completion of the reaction, the reaction mixture was dialyzed overnight against a Tris-HCI solution at 4℃and 20mM and pH8.0, freeze-dried and stored at-20 ℃.
5) 2ML of the complex was used to detect IGF-1 and IGFBP-3 by radioimmunoassay.
The results showed that the IGF-1 concentration in the complex was 536.45mg/ml and IGFBP-3 concentration was 158.36mg/ml.
Example 4 detection of IGF-1 biological Activity in Complex fluid
The recovered NIH3T3 cells were cultured in DMEM medium containing 10% calf serum, and after 80% growth, they were transferred to 6-well plates and cultured for 24 hours. NIH3T3 cells were starved with DMEM medium containing 2% calf serum for 24h, and IGF-1 recombinant protein was added to final concentrations of 0, 100, 200, 400ng/ml, respectively. After further culturing for 24 hours, 48 hours and 72 hours, the proliferation of the cells was detected by MTT method.
MTT assay showed an increase in proliferation rate of NIH3T3 cells following addition of IGF-1 at various concentrations. Cell cycle results from flow cytometry showed that after 48h of NIH3T3 cells treated with IGF-1 (at a concentration of 200 ng/ml), the percentage of cells in the G1 and G0 phases was reduced from 79.0% to 51.4% (P < 0.05) and the percentage of cells in the S phase was increased from 14.1% to 36.7% (P < 0.05) compared to the control group without IGF-1 treatment.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Suzhou Hongguanzhuang national medicine Co., ltd
<120> A method for preparing a fusion protein of IGFBP-3 and IGF-1 and a complex
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 335
<212> PRT
<213> Fusion protein (IGFBP-3-Glu-IGF-1)
<400> 1
Gly Ala Ser Ser Ala Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys
1 5 10 15
Asp Ala Arg Ala Leu Ala Gln Cys Ala Pro Pro Pro Ala Val Cys Ala
20 25 30
Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu
35 40 45
Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly
50 55 60
Leu Arg Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu
65 70 75 80
Leu Asp Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu
85 90 95
Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu
100 105 110
Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser
115 120 125
Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser Lys
130 135 140
Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys
145 150 155 160
Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu
165 170 175
Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp
180 185 190
Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val
195 200 205
His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
210 215 220
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys
225 230 235 240
Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val
245 250 255
His Cys Tyr Ser Met Gln Ser Lys Glu Gly Pro Glu Thr Leu Cys Gly
260 265 270
Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe
275 280 285
Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro
290 295 300
Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg
305 310 315 320
Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala
325 330 335
Claims (4)
1. A fusion protein comprising IGFBP-3 and IGF-1, wherein said fusion protein comprises IGFBP-3 and IGF-1 and glutamic acid, said glutamic acid being located at a dipeptide bond forming a linkage between IGFBP-3 and IGF-1;
The IGFBP3, glutamic acid and IGF1 are sequentially connected to form the fusion protein, the tail end amino acid of the IGFBP-3 is lysine, the head end amino acid of the IGF-1 is glycine, the side chain structure H 2N-(CH2)4 -of the lysine and the side chain structure HOOC- (CH 2)2 -of the glutamic acid form an amide bond-CO-NH 2 through condensation, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
2. A method for preparing IGFBP-3 and IGF-1 complexes, wherein the fusion protein of claim 1 is cleaved in hydroxylamine solution.
3. The method for preparing IGFBP-3 and IGF-1 complex according to claim 2, the method is characterized in that the cleavage conditions of the fusion protein are as follows: hydroxylamine concentration was 3m, ph was 9.0, reaction temperature was 40 ℃, and reaction time was 8h.
4. An engineering bacterium, characterized in that the engineering bacterium comprises the gene of the fusion protein of claim 1.
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