Thrombin-antithrombin compound mutant with improved stability and application thereof

Abstract

The invention discloses a stability-improved thrombin-antithrombin compound mutant as well as a stably transfected cell and application thereof. The mutant is prepared by mutating proline at the 198 site of a thrombin B chain as shown in an amino acid sequence SEQ ID NO.1 into phenylalanine, the thrombin-antithrombin compound mutant is obtained by mutating asparagine at the 217th site of antithrombin as shown in SEQ ID NO.3 into phenylalanine, aspartic acid at the 327th site of the antithrombin as shown in SEQ ID NO.3 into isoleucine and glutamic acid at the 414th site of the antithrombin as shown in SEQ ID NO.3 into tyrosine, the stability of the thrombin-antithrombin compound mutant is remarkably improved, and the thrombin-antithrombin compound mutant is added into calf serum, so that the thrombin-antithrombin compound mutant is obtained. The thrombin-antithrombin composite material control product and the preparation method thereof solve the problems that the thrombin-antithrombin composite material control product for detecting thrombus diseases is poor in stability and large in resource consumption.

Description

Technical Field

[0001] The invention belongs to the field of gene engineering and protein engineering, and in particular relates to a thrombin-antithrombin complex mutant with improved stability, a stably transfected cell and an application thereof. Background Art

[0002] Thrombin is a marker that reflects the occurrence of coagulation. Its half-life is extremely short, only a few seconds, and it is difficult to measure directly. After the formation of thrombin, it quickly covalently binds with antithrombin (AT) to form a thrombin-antithrombin complex (TAT). The half-life in plasma is about 3 to 15 minutes. It can indirectly reflect the amount of thrombin generated and is a sign of the activation of the coagulation system. It can directly and sensitively reflect the degree of activation of the coagulation system, and further predict the formation of thrombus and the recurrence of re-thrombus at an early stage, which has important clinical significance.

[0003] At present, the main method for detecting TAT is chemiluminescence. The only imported product on the market is the TAT detection kit produced by Sysmex Medical Electronics Co., Ltd., and similar products with relatively mature characteristics are scarce in the domestic market. As an important component of chemiluminescence reagents, quality control products are mainly used to evaluate and monitor the accuracy of the detection system. At the same time, they can also help laboratories identify potential problems and take corrective measures in a timely manner, which is of great significance for clinical testing. The core component of quality control products is thrombin-antithrombin complex protein, which is a heterotrimer with extremely complex spatial conformation. Thrombin is composed of two polypeptide chains, chain A contains 36 amino acid residues, and chain B contains 259 amino acid residues. The two chains are covalently connected by disulfide bonds and then covalently bound to antithrombin to form a thrombin-antithrombin complex. There is no commercial sale of pure natural thrombin-antithrombin complex. The content in serum is very small, not enough to be extracted. The protein has poor stability and is difficult to recombinantly express, which brings certain difficulties to its application.

[0004] The existing thrombin-antithrombin complex material controls on the market all have the problem of poor stability, which will directly lead to the degradation or denaturation of the target to be detected, and ultimately reduce the accuracy and stability of the detection results of the detection reagents. For example, the thrombin-antithrombin complex material control produced by Sysmex Medical Electronics Co., Ltd. (Registration Certificate No.: Guoxie Injection 20162404951) has a validity period of 9 months and can only be used on the same day after opening the bottle and re-dissolving. The invention patent with application number 202410069367.2 discloses a method for preparing a TAT quality control product and its application, wherein porcine thrombin is added to porcine plasma, and thrombin combines with antithrombin in the plasma to form a thrombin-antithrombin complex, which is then prepared and freeze-dried to obtain a TAT quality control product. However, the porcine thrombin-antithrombin complex is quite different from the human thrombin-antithrombin complex, and its specificity and consistency for human medical testing are difficult to guarantee.

[0005] In view of this, it is of great significance to develop human thrombin-antithrombin complex proteins or their expression cell lines with good stability and potential application value, as well as quality control products for detecting thrombotic diseases. Summary of the invention

[0006] The present invention utilizes theoretical design based on crystal structure and protein folding free energy to perform molecular modification on thrombin B chain (amino acid sequence such as SEQ ID NO.1, PDB ID: 4H6T) and antithrombin (amino acid sequence such as SEQ ID NO.3, PDB ID: 1ANT), performs site-directed mutation on multiple amino acids, thereby obtaining a thrombin-antithrombin complex mutant with improved stability, and further provides a preparation method and application of a thrombin-antithrombin complex control product, thereby solving the problems of poor stability and high resource consumption of quality control products used to detect thrombotic diseases.

[0007] In one embodiment of the present invention, the mutant is obtained by mutating the proline at position 198 of the thrombin B chain as shown in SEQ ID NO.1 to phenylalanine, named P198F, and its amino acid sequence is shown in SEQ ID NO.4, and at the same time, the asparagine at position 217 of the antithrombin as shown in SEQ ID NO.3 is mutated to phenylalanine, the aspartic acid at position 327 is mutated to isoleucine, and the glutamic acid at position 414 is mutated to tyrosine, named N217F / D327I / E414Y, and its amino acid sequence is shown in SEQ ID NO.5.

[0008] The present invention also provides a gene encoding the above thrombin-antithrombin complex mutant.

[0009] The invention also provides a recombinant vector carrying the gene.

[0010] In one embodiment of the present invention, pCHO1.0 plasmid is used as the recombinant vector.

[0011] The present invention also provides a dihydrofolate reductase-deficient (DHFR-) Chinese hamster ovary cell (CHO-TAT-Clone 35). The CHO-TAT-Clone 35 cell line integrates the above gene or recombinant vector and can stably and efficiently express a thrombin-antithrombin complex mutant with improved stability.

[0012] The present invention also provides a method for preparing the above mutant, which specifically comprises the following steps: (1) transfecting CHODG44 cells with the above recombinant vector, and obtaining a recombinant cell line by screening with puromycin and methotrexate; (2) culturing the recombinant cell line, separating the supernatant by centrifugation, and affinity purifying to obtain a thrombin-antithrombin complex mutant.

[0013] The present invention also provides the use of the above-mentioned thrombin-antithrombin complex mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell line in the preparation of a thrombin-antithrombin complex substance control product.

[0014] The raw and auxiliary materials of the quality control product include: calf serum, 2% bovine serum albumin, 3% trehalose, 4% lactose, 3% polyethylene glycol, and 0.2% preservative. The thrombin-antithrombin complex control product is obtained by vacuum freeze-drying.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] (1) The CHO-TAT-Clone 35 cell line of the present invention can efficiently express a thrombin-antithrombin complex mutant with significantly improved stability, providing a more economical method for large-scale production of high-quality thrombin-antithrombin complexes;

[0017] (2) The thrombin-antithrombin complex material control product of the present invention has good stability, low cost, and is easy to use. It can be used for quality control of clinical testing of thrombin-antithrombin complexes to ensure the accuracy and reliability of detection, and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are mutation sites of the thrombin-antithrombin complex: P198F: the proline at position 198 of the B chain of thrombin mutates to phenylalanine; N217F: the asparagine at position 217 of antithrombin mutates to phenylalanine; D327I: the aspartic acid at position 327 of antithrombin mutates to isoleucine; E414Y: the glutamic acid at position 414 of antithrombin mutates to tyrosine;

[0019] Figure 2To select 10 stable cell batches for analysis of the expression of thrombin-antithrombin complex mutants;

[0020] Figure 3 This is the polyacrylamide gel electrophoresis analysis of the purified thrombin-antithrombin complex mutants, where 1 is non-denaturing electrophoresis and 2 is denaturing electrophoresis. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Experimental Materials and Reagents

[0023] 1. Host cells and vectors: CHO DG44 cells were purchased from Thermo Scientific, and pCHO1.0 vector was purchased from Beijing Huayueyang Biotechnology.

[0024] 2. Endonucleases and other biochemical reagents: Restriction endonucleases were purchased from NEB, AvrII endonuclease catalog number R0174S, BstZ17I endonuclease catalog number R3594S, EcoRV endonuclease catalog number R0195S, PacI endonuclease catalog number R0547S, ClonExpress II One Step Cloning Kit was purchased from Nanjing Novozymes Biotech Co., Ltd., and puromycin hydrochloride and methotrexate were purchased from Sangon Biotech Co., Ltd.

[0025] 3. Culture medium: CD DG44 culture medium was purchased from Thermo Scientific.

[0026] 4. Kit: Thrombin-antithrombin complex detection kit (chemiluminescence method) was purchased from Sysmex Medical Electronics Co., Ltd.

[0027] Example 1: Prediction and screening of mutation sites

[0028] The amino acid sequence and three-dimensional structure of each subunit of the thrombin-antithrombin complex were analyzed to predict potential stable mutation sites. The sites located on the subunit contact surface and excluding the sites involved in the intermolecular interaction were selected. The folding energy change value ΔΔG of each amino acid site replaced by other amino acids was analyzed using PoPMuSiC-2.1 (Table 1). Finally, a mutant with four important stable mutation sites was obtained, named P198F / N217F / D327I / E414Y ( Figure 1 ).

[0029]

[0030]

[0031] Table 1 ΔΔG values ​​of thrombin-antithrombin complex subunit mutants

[0032] Example 2: Synthesis of mutant encoding gene and construction of recombinant vector

[0033] Without changing the amino acid sequence, the gene sequence encoding the above P198F / N217F / D327I / E414Y mutant was codon optimized to increase the expression in the host cell. The optimized nucleotide sequence of thrombin B chain (P198F) was such as SEQ ID NO.6, and AvrII and BstZ17I restriction enzyme sites were added to the 5' end and 3' end respectively, and a Strep tag was added to the 3' end at the same time. The optimized nucleotide sequence of thrombin A chain was such as SEQ ID NO.7, and EcoRV and PacI restriction enzyme sites were added to the 5' end and 3' end respectively, and a His tag was added to the 3' end at the same time. The optimized nucleotide sequence of antithrombin (N217F / D327I / E414Y) was such as SEQ ID NO.8, and AvrII and BstZ17I restriction enzyme sites were added to the 5' end and 3' end respectively, and a His tag was added to the 3' end at the same time, and artificial synthesis was performed.

[0034] According to the above synthesized gene sequence, primers were designed and synthesized (Table 2), and PCR amplification was performed using the synthesized gene as a template. The PCR reaction parameters were: denaturation at 94°C for 5min; then denaturation at 94°C for 30sec, annealing at 45°C for 30sec, extension at 72°C for 1min, and insulation at 72°C for 10min after 30 cycles. The thrombin B chain, A chain and antithrombin gene fragments were obtained, and the recovered fragments were connected to the pCHO1.0 vector using the ClonExpress IIOneStep Cloning Kit, where the thrombin B chain and A chain were connected to the same vector, named pCHO1.0-Chain B-Chain A, and the antithrombin was connected to another vector, named pCHO1.0-Antithrombin. The constructed vector was sent to Sangon Biotech Co., Ltd. for sequencing, confirming that the mutations at the four positions of P198F / N217F / D327I / E414Y were correct.

[0035]

[0036]

[0037] Table 2 Primer sequence list

[0038] Example 3: Construction of CHO DG44 cells stably expressing thrombin-antithrombin complex mutants

[0039] Prepare CHO DG44 cells and endotoxin-free pCHO1.0-Chain B-Chain A and pCHO1.0-Antithrombin plasmids for transfection at a rate of 10 μg plasmid (5 μg each of pCHO1.0-Chain B-Chain A and pCHO1.0-Antithrombin) / 1×10 7 After transfection, the cells were transferred to a cell culture flask and placed in a 37°C, 5% carbon dioxide incubator for static culture. After 24 hours, the cells were resuspended in CD DG44 medium at 5×10 5The density of viable cells / mL was transferred into T25 cell culture flasks, and 10μg / mL puromycin and 100nM / L methotrexate were added to screen stably transfected cells. The cells were cultured at 37°C and 5% carbon dioxide for 10-15 days. Fresh CD DG44 medium containing 10μg / mL puromycin and 100nM / L methotrexate was replaced every 3-4 days. When the cell viability was greater than 80%, the concentrations of puromycin and methotrexate were increased to 20μg / mL and 200nM / L, respectively, for the second round of pressure screening. The second round of pressure screening lasted for about 3-4 weeks. When the cell viability was greater than 85%, the monoclonal cell lines were screened by limiting dilution method, and the expression of thrombin-antithrombin complex mutants of each monoclonal cell line was analyzed by indirect ELISA. Ten cell lines with higher expression were selected for batch culture, and 3×10 5 The density of viable cells / mL was inoculated in a 125mL cell shake flask and cultured continuously at 37°C, 5% carbon dioxide, and 120rpm constant temperature shaking shaker until the cell viability dropped below 60%. During this period, the cell growth and expression level were monitored daily. According to the cell number, viability, and expression level of the thrombin-antithrombin complex mutant (indirect ELISA), a cell line with stable growth and high expression level was screened and named CHO-TAT-Clone 35 ( Figure 2 ).

[0040] Example 4: Purification of Thrombin-Antithrombin Complex Mutants

[0041] The cell line obtained above that can stably and efficiently secrete the thrombin-antithrombin complex mutant was cultured at 5×10 5 The density of viable cells / mL was inoculated in a 1L cell shake flask and cultured continuously for 10-12 days at 37°C, 5% carbon dioxide, and 120rpm constant temperature shaking shaker. The supernatant was collected by centrifugation at 9000rpm to purify the target protein.

[0042] The Ni Bestarose FF chromatography column (Borgron, AA0052) was washed with 10 mmol PBS equilibration buffer until the pH, conductivity and UV of the effluent were the same as those of the equilibration buffer, the collected supernatant was slowly loaded, the chromatography column was washed with equilibration buffer until the UV absorbance was close to the baseline, the target protein was eluted with 10 mmol PBS buffer containing 500 mmol / L imidazole, and dialyzed into 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0 binding buffer, and then loaded onto a Strep-Tactin Berpharose FF chromatography column (Borsi, BTR211Q), and finally the target protein was eluted with a binding buffer containing 2.5 mM dethiobiotin, and dialyzed into 10 mmol PBS buffer, and then passed through Bestdex G-25 gel filtration medium (Borgron, AG0132) to collect high-purity thrombin-antithrombin complex mutants ( Figure 3 ).

[0043] Example 5: Application of thrombin-antithrombin complex mutants

[0044] Commercial calf serum was selected, 2% bovine serum albumin, 3% trehalose, 4% lactose, 3% polyethylene glycol and 0.2% preservative were added, and the above-mentioned purified thrombin-antithrombin complex mutant was added, and the concentrations were 2ng / mL, 20ng / mL and 80ng / mL, respectively, and the natural protein thrombin-antithrombin complex was used as a control. The prepared substances were divided into 3mL freeze-drying bottles according to the specification of 1.0mL / bottle for vacuum freeze drying (vacuum degree <5Pa, cold trap temperature <-55℃, -50℃ pre-freezing for 2 hours, and heating and drying at 5℃ every 2 hours until 25℃), and three levels (level 1: 2ng / mL, level 2: 20ng / mL, level 3: 80ng / mL) of thrombin-antithrombin complex material control products were obtained, and the covers were placed at 2-8℃ for storage.

[0045] The thrombin-antithrombin complex detection kit (chemiluminescence method) of Sysmex Medical Electronics Co., Ltd. was used to test the reconstituted refrigerated stability, reconstituted room temperature stability, reconstituted frozen storage stability and 37°C accelerated stability of the above two thrombin-antithrombin complex control products.

[0046] (1) Evaluation of the stability of the control product after reconstitution at 2-8°C

[0047] The two quality control products were reconstituted with 1.0 mL of purified water and stored at 2-8°C. Sampling comparison tests were performed at 4 days, 8 days, 12 days, and 16 days, respectively. Compared with the freshly reconstituted quality control product, the stability of the thrombin-antithrombin complex control product of the present invention was significantly better than that of the natural protein thrombin-antithrombin complex control product. The data are shown in Table 3.

[0048]

[0049] Table 3 Evaluation of storage stability at 2-8℃ after reconstitution

[0050] (2) Evaluation of the stability of quality control products after reconstitution and storage at room temperature

[0051] The two quality control products were reconstituted with 1.0 mL of purified water and placed at room temperature for 6 hours. Sampling comparison tests were performed every two hours. Compared with the freshly reconstituted quality control products, the stability of the thrombin-antithrombin complex control product of the present invention was significantly better than that of the natural protein thrombin-antithrombin complex control product. The data are shown in Table 4.

[0052]

[0053]

[0054] Table 4 Evaluation of stability of storage at room temperature after reconstitution

[0055] (3) Evaluation of the stability of quality control products after reconstitution and cryopreservation

[0056] The two quality control products were reconstituted with 1.0 mL of purified water and packaged, stored at -20°C, and compared and tested at 7 days, 14 days, 21 days, and 28 days, respectively. Compared with the freshly reconstituted quality control product, the stability of the thrombin-antithrombin complex control product of the present invention is significantly better than the stability of the natural protein thrombin-antithrombin complex control product. The data are shown in Table 5.

[0057]

[0058] Table 5 Evaluation of stability of repackaged frozen storage after reconstitution

[0059] (4) Evaluation of the storage stability of quality control products at 37°C

[0060] The two quality control products were stored at 37°C and compared at 1 day, 5 days, 9 days and 13 days, and compared with the freshly reconstituted quality control product. The stability of the thrombin-antithrombin complex control product of the present invention is significantly better than that of the natural protein thrombin-antithrombin complex control product. The data are shown in Table 6.

[0061]

[0062] Table 6 Evaluation of storage stability at 37°C

[0063] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A thrombin-antithrombin complex mutant, characterized in that: The mutant is obtained by mutating the proline at position 198 of the thrombin B chain shown in the amino acid sequence SEQ ID NO.1 to phenylalanine, and simultaneously mutating the asparagine at position 217 of the antithrombin shown in SEQ ID NO.3 to phenylalanine, the aspartic acid at position 327 to isoleucine, and the glutamic acid at position 414 to tyrosine.

2. A gene encoding the thrombin-antithrombin complex mutant according to claim 1.

3. The gene of the thrombin-antithrombin complex mutant according to claim 2, characterized in that The gene sequences are as follows: SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.

8.

4. A recombinant vector carrying the gene according to claim 2 or claim 3.

5. A stably transfected cell according to claim 2, 3 or 4.

6. A method for preparing a thrombin-antithrombin complex mutant based on the recombinant vector according to claim 4, characterized in that: The following steps are involved: (1) transfecting the recombinant vector into CHO DG44 cells, and selecting the recombinant cell strain by puromycin and methotrexate; (2) Cultivating the recombinant cell line, collecting the supernatant by centrifugation, and obtaining the thrombin-antithrombin complex mutant by step-by-step affinity purification.

7. Use of the thrombin-antithrombin complex mutant according to claim 1 in the preparation of a thrombin-antithrombin complex material control product.

8. Use of the gene according to claim 2 or 3 in the preparation of a thrombin-antithrombin complex control product.

9. Use of the recombinant vector according to claim 4 in the preparation of a thrombin-antithrombin complex control product.

10. Use of the stably transfected cells according to claim 5 in the preparation of a thrombin-antithrombin complex control product.

11. A method for preparing a thrombin-antithrombin complex control product, characterized in that: The thrombin-antithrombin complex mutant according to claim 1 or the culture of the stably transfected cells according to claim 5 is added to calf serum, and 2% bovine serum albumin, 3% trehalose, 4% lactose, 3% polyethylene glycol, and 0.2% preservative are added at the same time, and the mixture is prepared by vacuum freeze-drying.

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