Recombinant expression vector and cell for producing bFGF and production method

Abstract

The invention discloses a recombinant expression vector and cell for producing bFGF and a production method, and relates to the technical field of biological protein production. According to the production method, a recombinant expression vector for highly expressing the bFGF is prepared by improvement on the basis of a pGEX-6P-1 commercial vector, escherichia coli cells are transformed by the recombinant expression vector to obtain recombinant escherichia coli cells, and then the high-yield and high-purity bFGF is obtained by utilizing the recombinant escherichia coli cells. The technical bottlenecks of low expression efficiency, complex purification process, high cost, poor vector adaptability and the like in the existing recombinant bFGF production are remarkably solved through promoter modification, fusion expression unit design and intein mutation optimization of an expression vector.

Description

Technical Field

[0001] The present invention relates to the technical field of biological protein production, and in particular to a recombinant expression vector and cell for producing bFGF and a production method. Background Art

[0002] Basic fibroblast growth factor (bFGF) is a cytokine with a wide range of biological activities and has important applications in wound repair, neural regeneration, tissue engineering, and other fields. Currently, the production of recombinant bFGF mainly relies on the Escherichia coli expression system, which has advantages such as low material costs, high process maturity, and great scalability potential. However, the existing technology of E. coli expression of bFGF still faces multiple technical bottlenecks, which limits the efficiency of industrialization:

[0003] 1. Insufficient expression efficiency and solubility

[0004] In conventional expression systems, over 50% of bFGF product accumulates as inactive inclusion bodies due to differences in codon preference, a lack of molecular chaperones, and an insufficient oxidative folding environment. Unoptimized bFGF yields typically fall below 2 mg / L, requiring tedious refolding steps such as gradient dialysis and redox buffer treatment to restore activity, resulting in process losses of up to 60-80%.

[0005] 2. The purification process is complex and costly

[0006] Traditional methods require a multi-step (6-8) purification process involving nickel affinity chromatography, ion exchange, and molecular sieve separation. This is not only cumbersome and requires significant equipment investment, but also results in low product recovery. For example, using a basic vector like pET28a, the overall production cost of bFGF can reach 5,000-6,000 yuan per microgram, severely restricting both scientific research and clinical application.

[0007] 3. Difficulties in regulating intein self-cleavage

[0008] While purification strategies based on self-cleavage of inteins (such as Mxe GyrA) can simplify the process, wild-type inteins are subject to issues such as nonspecific cleavage and precursor protein instability. For example, unmutated Mxe GyrA inteins are prone to premature self-cleavage during expression, resulting in reduced target protein recovery and the reliance on expensive affinity media (such as chitin) for purification.

[0009] 4. Poor carrier adaptability

[0010] Wang Hao et al. disclosed a mutant Mxe GyrA intein technology in "A practical approach to unveiling auto-catalytic cleavagesmediated by Mxe GyrA intein and improving the production of authentic bFGF". Although this technology solves the problem of premature self-cleavage during expression, it is only applicable to specific unconventional vectors, such as pWK3R-derived vectors, and is difficult to apply to commercial vectors, resulting in limited production prospects and poor compatibility with commercial production systems.

[0011] Therefore, developing a technical solution that can synergistically improve the expression level, solubility and purification efficiency of bFGF and utilize commercial vectors is the key to breaking through the bottleneck of the existing Escherichia coli expression system and is of great significance to promoting the industrial application of bFGF. Summary of the Invention

[0012] In order to solve the technical problem of high production costs in existing technologies, the present invention provides a new solution, namely, improving the commercial pGEX-6P-1 vector to prepare a recombinant expression vector that highly expresses bFGF, and using the vector to transform Escherichia coli cells to obtain recombinant Escherichia coli cells, and then using the recombinant Escherichia coli cells to obtain a high-yield, high-purity bFGF production method.

[0013] The technical solution adopted in the present invention is as follows:

[0014] A recombinant expression vector for producing bFGF comprises a CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit and a pGEX-LacUV5 modified vector of a pGEX-6P-1 vector, wherein the pGEX-LacUV5 modified vector is obtained by replacing the tac promoter of the pGEX-6P-1 vector with a LacUV5 promoter through cloning technology, and the CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit is loaded into the pGEX-LacUV5 modified vector.

[0015] Furthermore, the nucleotide sequence of the LacUV5 promoter is shown in SEQ ID NO: 1.

[0016] SEQ ID NO:1TTTCAAATCGATCGATCGATCGAT

[0017] Furthermore, the amino acid sequence of the CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit is shown in SEQ ID NO:2.

[0018] SEQ ID NO:2MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF *.

[0019] Furthermore, the CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit was double-digested with SalI and XbaI and then inserted into the pGEX-LacUV5 transformation vector.

[0020] A recombinant Escherichia coli cell is obtained by transforming an Escherichia coli cell with the recombinant expression vector described in any one of the above schemes.

[0021] More preferably, the Escherichia coli is a BL21DE3 strain.

[0022] A method for producing bFGF comprises the following steps:

[0023] (1) culturing the recombinant Escherichia coli cells according to claim 5 or 6, and inducing the expression of the fusion protein by IPTG;

[0024] (2) collecting and lysing the bacterial cells to obtain a cell lysate containing the CBD-MxeGyrA(C1A)-bFGF-his fusion protein;

[0025] (3) bFGF can be obtained by nickel column purification.

[0026] In summary, compared with the existing technology, the present invention significantly solves the technical bottlenecks of low expression efficiency, complex purification process, high cost, and poor vector adaptability in the existing recombinant bFGF production through promoter modification of the expression vector, design of fusion expression unit, and optimization of intein mutation. The advantages and beneficial effects are analyzed as follows:

[0027] 1. Significantly improve expression and solubility, breaking through the bottleneck of low yield

[0028] By replacing the strong Tac promoter in the pGEX-6P-1 vector with the weak LacUV5 promoter, the single mutant CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit can be combined to significantly increase expression levels, improving the vector's adaptability. Furthermore, the weak LacUV5 promoter reduces the metabolic burden on host cells, avoiding plasmid instability and protein misfolding caused by strong promoters. Experimental data showed that the bFGF yield of the recombinant bFGF fusion unit in the pGEX-LacUV5 modified vector recombinant plasmid increased to 96 mg / L, and the soluble fraction was greatly improved, significantly reducing inclusion body formation and eliminating the complex renaturation step.

[0029] 2. Optimize the self-cleavage efficiency of intein and simplify the purification process

[0030] The introduction of an Mxe GyrA intein mutant (C1A) and a his tag solves the problems of nonspecific shearing of the wild-type intein and instability of the precursor protein. The combination of the pGEX-LacUV5 modified vector of the present invention with the Mxe GyrA intein mutant (C1A) and the his tag significantly improves the self-cleavage efficiency, enabling sufficient self-cleavage within the bacteria. After cell lysis, the lysate is collected and bFGF can be directly purified using a nickel column without inducing shearing in vitro. This significantly shortens the purification steps, significantly improves the recovery rate, and significantly reduces operational complexity and equipment investment.

[0031] 3. Enhance the compatibility between promoter and vector and improve system stability

[0032] The conserved sequence in the -10 region of the LacUV5 promoter has a higher affinity for E. coli RNA polymerase and is independent of CAP protein regulation, resulting in more rigorous transcription initiation. Its compatibility with the pGEX-6P-1 vector backbone (containing the ori replicon and ampR resistance gene) is significantly better than that of the Tac promoter. In the absence of induction, basal expression is reduced, preventing damage to the host by toxic proteins and significantly improving cell survival.

[0033] 4. Reduce production costs and have industrial advantages

[0034] Since the expression level is increased by 60 times and the purification steps are reduced by more than 50%, the overall production cost of recombinant bFGF is greatly reduced.

[0035] 5. Maintain high activity of target protein to meet clinical application needs

[0036] Through weak promoter regulation and precise intein cleavage, protein aggregation and activity loss caused by overexpression are avoided. MTT assay verification showed that the purified recombinant bFGF exhibited comparable proliferation-promoting activity (EC50 = 0.1 ng / mL) on BALB / 3T3 cells to that of the native protein. With a purity of 98%, it meets biopharmaceutical-grade product standards and can be directly applied in clinical scenarios such as wound repair and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : This is a gel image of the induced bacterial fission liquid obtained in the experimental group and the control group 1 in Example 1;

[0038] Figure 2 This is a graph showing the biological activity test results of the bFGF prepared in Example 3 and commercially available bFGF, wherein the solid line represents the bFGF prepared in Example 3, and the dotted line represents the commercially available bFGF. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific embodiments, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0040] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] The present application will be described in detail below with reference to specific embodiments and experimental data.

[0043] Example 1

[0044] In this example, a recombinant expression vector was constructed and expression verification was performed. The specific steps and results are as follows:

[0045] 1. Construction of pGEX-LacUV5 modified vector: Using pGEX-6P-1 as the original vector, replace the Tac promoter with the LacUV5 promoter.

[0046] 2. Construction of recombinant expression vector:

[0047] (1) Experimental group: The CBD-MxeGyrA (C1A)-bFGF-his fusion expression unit containing the mutant intein (C1A) and bFGF gene sequence was inserted into the pGEX-LacUV5 modified vector to construct the recombinant vector pLacUV5-CBD-MxeGyrA (C1A)-bFGF-his;

[0048] (2) Control group 1: The CBD-MxeGyrA (C1A)-bFGF-his fusion expression unit containing the mutant intein (C1A) and bFGF gene sequence was inserted into the original pGEX-6P-1 vector to construct the recombinant vector pGEX-6P-1-CBD-MxeGyrA (C1A)-bFGF-his;

[0049] 3. Expression verification: The two recombinant vectors were transformed into E. coli BL21DE3, induced at 37°C with 0.1 mM IPTG for 4 h. Using the standard sample as a control, the induced bacterial lysates obtained from the experimental group and the control group 1 were subjected to electrophoresis. Figure 1 As shown, the experimental group produced more target product with fewer impurities, while the control group 1 had less target product, with almost no target product band visible. SDS-PAGE electrophoresis and BCA assay were used to determine the fusion protein yield, soluble fraction, and inclusion body ratio. The results are shown in Table 1.

[0050] Table 1 Results of fusion protein yield, soluble ratio and inclusion body ratio of different vectors

[0051] Carrier type bFGF production (mg / L) Soluble ratio Inclusion body ratio Control group 1: pGEX-6P-1 ＜1 / / Experimental group: pGEX-LacUV5 96 92％ 8％

[0052] It can be seen that after the promoter improvement of the original pGEX-6P-1 vector, the bFGF production and solubility ratio can be increased and the inclusion body ratio can be reduced.

[0053] Example 2

[0054] The purpose of this example is to evaluate the compatibility of the LacUV5 promoter with the pGEX-6P-1 vector backbone and the system stability.

[0055] The specific experiments and results are as follows:

[0056] 1. Promoter affinity analysis: EMSA experiments were performed to test the binding ability of the -10 region of the LacUV5 promoter to the E. coli RNA polymerase σ70 factor. The results showed that the affinity of the LacUV5 promoter for RNA polymerase was 2.3 times higher than that of the Tac promoter, and the background expression was reduced by 90% in the absence of IPTG induction.

[0057] 2. Background expression and cell survival assays: lacI gene transcription levels were measured by qPCR under non-induction conditions, and cell survival was assessed by flow cytometry. Results: After 10 serial passages of the recombinant bacteria, the plasmid loss rate decreased from 28% to 5%, and cell survival increased from 62% to 95%.

[0058] It can be seen that the LacUV5 promoter has excellent compatibility with the pGEX-6P-1 vector, and the system stability is significantly improved.

[0059] Example 3

[0060] The purpose of this example is to verify the proliferation-promoting activity and purity of the recombinant bFGF prepared by the method of the present invention. The preparation and related determination methods and steps of the recombinant bFGF are as follows:

[0061] 1. The preparation process of the target for detection is as follows:

[0062] (1) The recombinant E. coli BL21DE3 prepared in Example 1 was cultured in 200 ml LB at 37°C until OD600nm = 1-2, and induced with 0.1 mM IPTG for 10 h;

[0063] (2) collecting and lysing the bacterial cells to obtain a cell lysate containing the CBD-MxeGyrA(C1A)-bFGF fusion protein;

[0064] (3) Using a general nickel column purification method, bFGF that is automatically cleaved by the intein inside the bacteria is purified from the supernatant of the bacterial lysate.

[0065] 2. Proliferation Activity Detection

[0066] (1) HPLC purity analysis: Reverse-phase high performance liquid chromatography was used to detect the purity of the target protein. The results showed that the bFGF obtained by HPLC detection had a purity of 98.3% and an activity retention rate of 92% after storage at 4°C for 30 days, which met the biological product standards of the "Chinese Pharmacopoeia".

[0067] (2) The purified bFGF was applied to BALB / 3T3 cells to determine the EC50 value. Commercially available bFGF was used as a control group. The results were as follows: Figure 2 As shown, the EC50 concentration of the recombinant bFGF prepared in this example on BALB / 3T3 cells had no statistical difference with that of the natural bFGF standard (p>0.05).

[0068] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0069] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, all of which fall within the scope of protection of the present application.

Claims

1. A recombinant expression vector for producing bFGF, comprising a CBD-MxeGyrA (C1A)-bFGF-his fusion expression unit, characterized in that: It also includes a pGEX-LacUV5 modified vector of the pGEX-6P-1 vector, wherein the pGEX-LacUV5 modified vector is obtained by replacing the tac promoter of the pGEX-6P-1 vector with the LacUV5 promoter through cloning technology, and the CB D-MxeGyrA (C1A)-bFGF-his fusion expression unit is loaded into the pGEX-LacUV5 modified vector.

2. The recombinant expression vector according to claim 1, wherein The nucleotide sequence of the LacUV5 promoter is shown in SEQ ID NO:

1.

3. The recombinant expression vector according to claim 1, wherein The amino acid sequence of the CBD-MxeGyrA(C1A)-bFGF-his fusion expression unit is shown in SEQ ID NO:

2.

4. The recombinant expression vector according to claim 1, wherein The CBD-MxeGyrA (C1A)-bFGF-his fusion expression unit is double-digested with SalI and XbaI and then inserted into the pGEX-LacUV5 transformation vector.

5. A recombinant Escherichia coli cell, characterized in that The recombinant Escherichia coli cell is obtained by transforming the recombinant expression vector according to any one of claims 1 to 4 into an Escherichia coli cell.

6. The recombinant Escherichia coli cell according to claim 5, wherein The Escherichia coli is BL21DE3 strain.

7. A method for producing bFGF, characterized in that: The steps include: (1) culturing the recombinant Escherichia coli cells according to claim 5 or 6, and inducing the expression of the fusion protein by IPTG; (2) collecting and lysing the bacterial cells to obtain a cell lysate containing the CBD-MxeGyrA(C1A)-bFGF-his fusion protein; (3) bFGF can be obtained by nickel column purification.

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