Anti-ATCA2 recombinant monoclonal antibody variable region coding gene and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a variable region coding gene of an anti-ATCA2 recombinant monoclonal antibody and a preparation method of the variable region coding gene. The variable region coding gene of the anti-ATCA2 recombinant monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises any one of amino acid sequences as shown in SEQ ID NO.6-9; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO. 10. According to the present invention, the mouse anti-human ACTA2 recombinant monoclonal antibody is prepared by using the hybridoma technology recombinant monoclonal antibody, and the recombinant monoclonal antibody has characteristics of clear structure, high titer and good stability, can effectively solve the problems existing in the traditional antibody preparation, can be used in the detection and screening fields such as immunohistochemistry, indirect ELISA, antibody chip preparation, immunofluorescence, and the like, and further has characteristics of high sensitivity, high sensitivity, and the like. And accurate evaluation and detection results can be obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant monoclonal antibody variable region encoding gene against ATCA2 and a preparation method thereof. Background Art

[0002] Antibodies refer to proteins with protective effects produced by the body due to the stimulation of antigens. They are a class of immunoglobulins that can specifically bind to antigens. ACTA2 (Actin, aortic smooth muscle, ACTA2) is a G protein-coupled receptor (GPCR), a dimer composed of an α subunit and a β subunit. ACTA2 plays a key role in controlling cardiovascular movement and maintaining blood pressure. In the heart, ACTA2 is located in the intracellular region of the cardiac β1 receptor and is a ligand-binding receptor for the cardiac β1 receptor. The cardiac β1 receptor is a GPCR located on the membrane of cardiac muscle cells. When the β1 receptor binds to ACTA2, it causes contraction and relaxation of cardiac muscle cells, thereby controlling heart rate and blood pressure. ACTA2 can also be used as a biomarker to detect the progression of cardiovascular disease and the effectiveness of treatment. Studies have shown that ACTA2 can regulate the expression of mesenchymal to epithelial transition factor (MET) and focal adhesion kinase (FAK) in lung adenocarcinoma cells, so ACTA2 may be a promising prognostic biomarker and anticancer drug target for metastatic lung adenocarcinoma.

[0003] Traditional antibody preparation generally obtains monoclonal antibodies and polyclonal antibodies by immunizing animals. Monoclonal antibodies are prepared by immunizing host animals with target proteins, extracting B cells that can recognize and respond to the antigen, and fusing them with myeloma cells to form hybridoma cells. Polyclonal antibodies are prepared by collecting blood from animals that have been immunized with target antigens. However, there are many problems with the antibodies currently prepared, such as low antibody titer, the purchased antibodies used to detect protein X preferentially bind to protein Y, or even do not bind to X at all; the antibody stability is poor, the batch differences are large, and the experimental results cannot be repeated when repeating previous experiments with different batches of antibodies, and the repeatability is poor.

[0004] The above problems have led to erroneous scientific discoveries and wasted scientific research efforts, causing huge losses in cancer, metabolism, aging, immunology and cell signal transduction, as well as any field of complex biological molecules. Therefore, it is of great significance to develop a method for preparing an anti-ACTA2 antibody with a clear structure, high titer and high stability. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a recombinant monoclonal antibody variable region encoding gene against ATCA2, a preparation method and an application thereof, and prepares a mouse anti-human ACTA2 recombinant monoclonal antibody by utilizing hybridoma technology to recombinant monoclonal antibodies. The recombinant antibody has a clear structure, high titer and better stability.

[0006] The first aspect of the present invention aims to provide a gene encoding the variable region of a recombinant monoclonal antibody against ATCA2.

[0007] The second aspect of the present invention aims to provide a nucleic acid.

[0008] The third aspect of the present invention is to provide a recombinant expression vector.

[0009] The fourth aspect of the present invention aims to provide a recombinant expression cell.

[0010] The fifth aspect of the present invention is to provide a method for preparing a gene encoding the variable region of a recombinant monoclonal antibody against ATCA2.

[0011] The object of the sixth aspect of the present invention is to provide a use of the nucleic acid described in the second aspect, or the recombinant monoclonal antibody variable region encoding gene against ATCA2 described in the first aspect, or the recombinant expression vector described in the third aspect, or the recombinant expression cell described in the fourth aspect in the preparation of a recombinant monoclonal antibody variable region encoding gene against ATCA2.

[0012] The object of the seventh aspect of the present invention is to provide an application of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 described in the first aspect, or the nucleic acid described in the second aspect, or the recombinant expression vector described in the third aspect, or the recombinant expression cell described in the fourth aspect, or the preparation method described in the fifth aspect in detecting ACTA2 protein molecules, or preparing a device for detecting ACTA2 protein molecules.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is: In a first aspect of the present invention, a recombinant monoclonal antibody variable region encoding gene against ATCA2 is provided, wherein the recombinant monoclonal antibody variable region encoding gene against ATCA2 comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO.6-9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.10.

[0014] Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.6, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.10; Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.10; Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.8, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.10; Furthermore, in the above-mentioned anti-ATCA2 recombinant monoclonal antibody variable region encoding gene, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.9, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.10.

[0015] The second aspect of the present invention provides a nucleic acid encoding the heavy chain variable region and the light chain variable region of the above-mentioned anti-ATCA2 recombinant monoclonal antibody variable region encoding gene.

[0016] Furthermore, the nucleic acid encoding the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 comprises: The nucleotide sequence shown in SEQ ID NO.1 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.6 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.2 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.7 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.3 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.8 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.4 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.9 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.5 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.10 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2.

[0017] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid.

[0018] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the above nucleic acid.

[0019] The fourth aspect of the present invention provides a recombinant expression cell, which contains the nucleic acid encoding the variable region of the recombinant monoclonal antibody against ATCA2, or the recombinant expression vector.

[0020] The fifth aspect of the present invention provides a method for preparing a recombinant monoclonal antibody variable region encoding gene against ATCA2. The preparation method comprises: using the above-mentioned recombinant expression vector to transfect cells to obtain recombinant expression cells, culturing the recombinant expression cells obtained after transfection, collecting and purifying the supernatant to obtain the recombinant monoclonal antibody variable region encoding gene against ATCA2.

[0021] The sixth aspect of the present invention provides a use of the above-mentioned nucleic acid, or the above-mentioned anti-ATCA2 recombinant monoclonal antibody variable region encoding gene, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant expression cell in the preparation of the anti-ATCA2 recombinant monoclonal antibody variable region encoding gene.

[0022] The seventh aspect of the present invention provides an application of the above-mentioned anti-ATCA2 recombinant monoclonal antibody variable region encoding gene, or the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant expression cell, or the above-mentioned preparation method in detecting ACTA2 protein molecules, or preparing equipment for detecting ACTA2 protein molecules.

[0023] Furthermore, the detection of ACTA2 protein molecules includes immunohistochemistry and affinity detection methods.

[0024] Furthermore, the immunohistochemical detection includes: dewaxing, antigen repair, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, sealing and microscopic examination.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a recombinant monoclonal antibody variable region encoding gene against ATCA2, a preparation method and application thereof, and the recombinant monoclonal antibody variable region encoding gene against ATCA2 is prepared by utilizing hybridoma technology to recombinant monoclonal antibodies, and the obtained antibody has a clear structure, high titer and good stability. The antibody is highly specific and sensitive in combination with the ACTA2 protein molecule, and can specifically identify and detect the expression of the ACTA2 protein on tumor tissues, and is positively highly expressed when detecting the ACTA2 protein. Therefore, the antibody can be applied to detection and screening fields such as immunohistochemistry, indirect ELISA, antibody chip preparation, and immunofluorescence, which is conducive to obtaining accurate evaluation and detection results.

[0026] The invention provides an anti-ATCA2 recombinant monoclonal antibody variable region encoding gene and a preparation method and application thereof, which have the characteristics of good specificity, strong positive signal and the like, and can provide a reference for clinical prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is a process flow chart of the preparation and expression of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of the present invention; Figure 2 is a gel electrophoresis diagram of total RNA of hybridoma cells of the present invention; Figure 3 It is a map of the variable region encoding gene of the recombinant monoclonal antibody against ACTA2 of the present invention; Figure 4 This is a gene amplification diagram of the mouse anti-human ACTA2 full-length recombinant antibody of the present invention; Figure 5 This is a diagram of the mouse heavy chain constant region vector pFUSEss-CHIg-mG2B of the present invention; Figure 6 This is a diagram of the mouse light chain constant region vector pFUSE2ss-CLIg-mk of the present invention; Figure 7 It is the preparation result of ACTA2 protein of the present invention; Figure 8 This is a diagram showing the results of SDS-PAGE detection of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of the present invention; Fig. 9This is a diagram of the sequencing results of the optimal anti-ATCA2 recombinant monoclonal antibody variable region encoding gene sequence T508-A1T-VK1 / VH2 of the present invention; Fig.10 This is a graph showing the results of the pilot SDS-PAGE test of the optimal anti-ATCA2 recombinant monoclonal antibody variable region encoding gene sequence T508-A1T-VK1 / VH2 using the ExpiCHO-S™ Expression System; Fig.11 This is a graph showing the titer test results of the ACTA2 recombinant antibody of the present invention; Fig.12 is the affinity constant curve of the ACTA2 recombinant antibody of the present invention; Fig.13 It is a Western Blot identification diagram of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of the present invention; Fig.14 It is a diagram showing the results of immunohistochemical verification of the tissue sample of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of the present invention. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0030] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form a new or preferred technical solution.

[0031] The flow chart of the method for preparing a recombinant monoclonal antibody variable region encoding gene against ATCA2 of the present invention is as follows: Figure 1 shown.

[0032] The following is further described with reference to specific embodiments.

[0033] The present invention provides a method for preparing a mouse anti-human ACTA2 recombinant monoclonal antibody, which specifically comprises the steps shown in Examples 1-3.

[0034] Example 1: Mouse monoclonal antibody variable region gene fishing 1. Hybridoma cell RNA extraction and reverse transcription results Mouse spleen cells and SP2 / 0 (mouse myeloma cells) were fused to obtain hybridoma cells; total RNA of hybridoma cells was extracted by Trizol method, and clear 28S and 18S bands were observed by agarose gel electrophoresis (such as Figure 2The RNA concentration and purity were measured, and the result was D(260 nm) / D(280 nm) = 1.98, which can meet the requirements of this experiment.

[0035] 3) Using the total RNA prepared above as a template, reverse transcription was performed using the Tiangen FastKing One-Step RT-PCR Kit (Cat. No.: KR123) to synthesize cDNA. The amplification system and procedure are shown in Tables 1 and 2. The amplification system includes 5×FastKing-RT SuperMix 4 µL; total RNA 50ng-2 µg; RNase-Free ddH2O supplemented to 20 µL. The amplification reaction procedure includes first reacting at 42°C for 15min and then reacting at 95°C for 3min.

[0036] Table 1 RNA reverse transcription system Table 2 The program settings are as follows: 2. PCR amplification of mouse monoclonal antibody variable region Using the cDNA prepared in step 1 as a template, one mouse VH upstream degenerate primer and one VH downstream degenerate primer were used to amplify the full set of VH genes; two mouse VK upstream degenerate primers and two VK downstream degenerate primers were mixed in a certain ratio to amplify the full set of VK genes. The amplification system, program and primer sequences are shown in Tables 3, 4 and 5. The amplification system includes 25 µL of Buffer; 1 µL of Phanta Max Super-Fidelity DNA Polymerase; 20 µL of ddH2O; 1 µL of upstream degenerate primer mix; 1 µL of downstream degenerate primer mix; and 1 µL of template cDNA. The amplification reaction program includes: first react at 95°C for 3 minutes, then react at 95°C, 56°C, and 72°C for 30 seconds each, for a total of 30 cycles, and finally react at 72°C for 5 minutes.

[0037] The agarose gel electrophoresis results of the amplified products were as follows Figure 3 As shown, Figure A shows the heavy chain / light chain variable region gene PCR amplification: 1-6: heavy chain variable region amplification, 7-12: light chain variable region amplification: VH gene fragment length is about 350-400 bp, VK gene fragment length is about 350 bp, which is consistent with the target fragment length.

[0038] Table 3 PCR amplification system Table 4 PCR amplification program Table 5 Primer sequences 3. Purification of V region genes and ligation with T-vector Purify the PCR product in step 2 using Tiangen Agarose Gel DNA Recovery Kit (Cat. No. DP209), connect the VH gene and VK gene to the pGEM-T vector, and transform E. coli. The connection reaction system is shown in Table 6.1, including 1 µL of PCR product, 1 µL of pGEM-T vector, and 3 µL of ddH2O.

[0039] The specific method is as follows: (1) Pick a single colony from the plate that has been cultured at 37°C for 16-20 h, inoculate it into 5 mL of LB culture medium, place it in a 37°C shaker, set the speed to 220 rpm / min, and culture for 6-8 h; (2) Inoculate the above initial culture into three 500 mL LB conical flasks containing 200 mL LB culture medium with inoculation volumes of 160 µL, 180 µL, and 200 µL, respectively, and shake overnight at 18°C ​​and 180 rpm. (3) The next morning, measure the OD600 values ​​of the three flasks of culture every 45 minutes. When the OD600 value of the culture is 0.55, place the culture flask on ice for 10 minutes. (4) Divide 200 mL of culture into four 50 mL centrifuge tubes and centrifuge at 5000 rpm for 10 min at 4°C to collect the cells. (5) Pour off the culture medium, absorb the remaining liquid with absorbent paper, and then add 20 mL of pre-cooled Inoue transformation buffer to each tube to resuspend the bacterial pellet. (Be careful to resuspend gently, do not use an oscillator or pipetting to mix); (6) Place in a centrifuge, adjust the temperature to 4°C, the speed to 5000 rpm, and centrifuge for 10 min to collect the cells; (7) Pour off the culture medium, absorb the remaining liquid with absorbent paper, combine the four tubes, add 20 mL of pre-cooled Inoue transformation buffer and gently resuspend the precipitate; (8) Add 1.5 mL of DMSO solution, gently mix the bacterial suspension, place on ice for 10 min, quickly dispense the suspension into cooled sterile microcentrifuge tubes, seal the tubes tightly, immerse the tubes in liquid nitrogen to quickly freeze the competent cells, and store them in a -80°C refrigerator for later use; (9) Use a sterile 10 µL pipette tip to pick up a single white colony and place it in a new 1.5 mL EP tube. Add 1 mL of liquid LB medium containing ampicillin to the tube and place it in a double-layer constant temperature culture shaker at 220 rpm and 37°C for 3-6 h. Remove the bacterial solution and set aside. (10) Prepare the reaction system according to Tables 6.1 and 6.2, mix well, and place in a PCR instrument; Table 6.1 Ligation reaction system Table 6.2 Bacterial liquid PCR amplification system (11) The bacterial solution PCR amplification program settings are shown in Table 6.3: Table 6.3 Bacterial PCR amplification program (12) Three light chain and heavy chain variable region genes were selected from clones that were positive for bacterial liquid PCR and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The universal primer CMV-F was used for colony PCR, and the target fragment size was about 500 bp. 18 T clones were selected for the heavy chain, of which 3, 5, 8, 10, 13, 14, and 18 were correct; 21 T clones were selected for the light chain, of which 1, 3, 5, 8, 10, 13, 15, 18, 19, 20, and 21 (such as Figure 3 As shown, Figure B is the identification of T clone colonies in the heavy chain variable region, and lanes 1 to 18 represent 1-18 T clones in the heavy chain variable region, among which 3, 5, 8, 10, 13, 14, and 18 are correct; Figure C is the identification of T clone colonies in the light chain variable region, and lanes 1 to 21 represent 1-21 T clones in the light chain variable region, among which 1, 3, 5, 8, 10, 13, 15, 18, 19, 20, and 21 are correct). The 7 positive clones of the heavy chain with bright bands and the 11 positive clones of the light chain were sent to Qingke Company for sequencing.

[0040] 4. Full-length recombinant antibody gene amplification The 7 heavy chain T clones and 11 light chain T clones were analyzed using IMGT or QUEST online analysis software, and a total of 1 effective light chain and 4 effective heavy chains were screened out, named as: T508-A1T-ACTA2-VK1, T508-A1T-ACTA2-VH1, T508-A1T-ACTA2-VH2, T508-A1T-ACTA2-VH3, T508-A1T-ACTA2-VH4, and the results of one of the effective heavy chain T508-A1T-ACTA2-VH4 and light chain T508-A1T-ACTA2-VK1 were used as representative analysis. The light chain T508-A1T-ACTA2-VK1 gene was analyzed for validity and VDJ gene family. The full length of the functional light chain variable region is 334 bases, and the functional light chain belongs to Musmus IGKV3-10 01 F family, the V region match rate was 96.22%, and the J region match rate was 92.11%. The heavy chain T508-A1T-ACTA2-VH2 gene was analyzed for validity and VDJ gene family. The functional heavy chain variable region was 352 bases long, and the functional heavy chains all belonged to Musmus IGHV14-3 02 F family, the V region matching rate was 96.18%, and the J region was 87.50%. The specific domain divisions of the light chain variable region T508-A1T-ACTA2-VK1 and the heavy chain variable region T508-A1T-ACTA2-VH2 are shown in Tables 7 and 8, respectively. The coding genes of the CDR1, CDR2, and CDR3 domains of the light chain variable region T508-A1T-ACTA2-VK1 and the heavy chain variable region T508-A1T-ACTA2-VH2 are shown in Tables 9 and 10, respectively.

[0041] Table 7 T508-A1T-ACTA2-VK1 light chain domain division Table 8 T508-A1T-ACTA2-VH2 heavy chain domain division Table 9 Light chain variable region domains and encoding genes Table 10 Heavy chain variable region domains and encoding genes Example 2: Construction of mouse anti-human ACTA2 full-length recombinant antibody vector In order to construct mouse anti-human ACTA2 full-length recombinant antibody plasmid, one light chain sequence (T508-A1T-ACTA2-VK1) and four heavy chain sequences (T508-A1T-ACTA2-VH1, T508-A1T-ACTA2-VH2, T508-A1T-ACTA2-VH3, T508-A1T-ACTA2-VH4) obtained by screening in Example 1 were constructed into pFUSE2ss-CLIg-mk and pFUSEss-CHIg-mG2B vectors, respectively. PCR amplification primers were designed based on the upstream and downstream restriction sites of the vectors to amplify the full-length recombinant antibody gene. The results are as follows: Figure 4 As shown in Figure A, bands 1-6 are heavy chain variable region amplifications, and bands 7-12 are light chain variable region amplifications. It can be seen from the figure that the size of the ACTA2 heavy chain variable region gene fragment is about 400 bp, and the size of the light chain variable region gene fragment is about 350 bp. The bands are clear and their molecular sizes are consistent with the expected theoretical values.

[0042] Subsequently, in order to identify whether the heavy chain and light chain variable region genes of the mouse anti-human ACTA2 hybridoma monoclonal antibody were successfully constructed into the pFUSEss-CHIg-mG2B and pFUSE2ss-CLIg-mk vectors, respectively, hEF1-HTLV prom and SV40 universal primers were used to perform colony identification on 4 T clones of the heavy chain variable region and 5 T clones of the light chain variable region. The results are shown in the figure. Figure 4 As shown in Figure B (Figure B shows colony identification of clones 1-4 for the heavy chain variable region gene, and colony identification of clones 5-9 for the light chain variable region gene), the light chain variable region gene is about 750 bp, and the heavy chain variable region gene is about 1500 bp, which is consistent with the length of the target fragment.

[0043] The specific method is as follows: 1. Design PCR amplification primers based on the upstream and downstream restriction sites of the vector as shown in Table 11, and then use the heavy chain and light chain variable region genes that are correct and functional as templates confirmed by sequencing.

[0044] Table 11 PCR amplification primer sequences 2. The mouse heavy chain constant region vector pFUSEss-CHIg-mG2B ( Figure 5 ) and mouse light chain constant region vector pFUSE2ss-CLIg-mk ( Figure 6 ) as the backbone, the amplified products of the heavy chain and light chain constant regions of the above mouse antibodies were recovered by gel and connected to the mouse heavy chain constant region vector pFUSEss-CHIg-mG2B and the mouse light chain constant region vector pFUSE2ss-CLIg-mk respectively.

[0045] (1) Light chain constant region modification: The vector pFUSE2ss-CLIg-mk was double-digested with EcoRI and XhoI to obtain two bands of 3524 bp and 336 bp in size; the rat light chain Igk constant region gene was amplified, double-digested with EcoRI and XhoI, and then connected to the 3524 bp target band, and sequenced for identification and confirmation. The nucleotide sequence encoding the light chain variable region (T508-A1T-ACTA2-VK1) is as follows: GACATTCAGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGTCAGAGGGCCACCATATCCTGCAGAACCAGTGAAAGTGTTGATAGTTATGGCAGAAATTTTATGCATTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTC CTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTATTGTCAGCAAAATAATGAGGATCCGCTCACTTTCGGTTCTGGGACCAATCTGGAGCTGAAAC(SEQ ID NO.5).

[0046] According to the codon encoding rules, the amino acid sequence of the light chain variable region (T508-A1T-ACTA2-VK1) is: DIQLTQSPASLAVSLGQRATISCRTSESVDSYGRNFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPLTFGSGTNLELK (SEQ ID NO.10).

[0047] (2) Heavy chain constant region modification: The vector pFUSEss-CHIg-mG2B was double-digested with NheI and EcoRI; the mouse heavy chain IgG2B constant region gene was amplified, double-digested with NheI and EcoRI, and then connected to the target band, and sequenced for identification and confirmation.

[0048] The nucleotide sequence encoding the first heavy chain variable region (T508-A1T-ACTA2-VH1) is as follows: GAGGTCAAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAGGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGGACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCGTTATACCCACGGGCTACTTTGACTACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCAG (SEQ ID NO. 1); The nucleotide sequence encoding the second heavy chain variable region (T508-A1T-ACTA2-VH2) is as follows: CAGGTGAAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATGTGCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTTCGAATGATTATGCTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACATCAGACACATCCTCCAACACAGCCTACCTGCAGGTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGGTCTTTACTACGGTCGTAGCATTGACTACTGGGGCCAAGGCACCACTCTCACTGTCTCCTCAG (SEQ ID NO. 2); The nucleotide sequence encoding the third heavy chain variable region (T508-A1T-ACTA2-VH3) is as follows: GAGGTCAAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATGTGCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTTCGAATGATTATGCTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACATCAGACACATCCTCCAACACAGCCTACCTGCAGGTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGGTCTTTACTACGGTCGTAGCATTGACTACTGGGGCCAAGGCACCACTCTCACTGTCTCCTCAG (SEQ ID NO. 3); The nucleotide sequence encoding the fourth heavy chain variable region (T508-A1T-ACTA2-VH4) is as follows: CAGGTGCAGCTGCAGGAGTCTGGGACTGAGCTGGCAAGCCCTGGGGCTTCAGTGAAGTTGTCCTGTAAGGCTTCTGGCTACATCTTTTCTGACTATTGGATGCAGTGGGTAAAAGAGAGGCCTGGACAGGGTCTGGAATGGATTGGGACTATTTATCCTGGAGATGGTGATGCTCGATACAATCTGAAGTTCAAGAACAAGGCCGCTTTGACTGCAGATAAATCTTCCAATACAGCCTACATGTTGCTCAACAACTTGGCATCTGAAGACTCTGCGGTCTATTACTGTGCAAGATACTGGGATTACGACGTTGGGGGGTTTACTTACTGGGGCCAGGGGTCTCTGGTCACTGTCTCTGCAG (SEQ ID NO.4).

[0049] According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (T508-A1T-ACTA2-VH1) is: EVKLQQSGAELVKPGASVRLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEINPNNGRTNYNEKFKSKATLTVDKSSRTAYMQLSSLTSEDSAVYYCAVIPTGYFDYWGQGTTLTVSS(SEQ ID NO.6); The amino acid sequence of the second heavy chain variable region (T508-A1T-ACTA2-VH2) is: QVKLQQSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPSNDYAKYDPKFQGKATITSDTSSNTAYLQVSSLTSEDTAVYYCGLYYGRSIDYWGQGTTLTVSS(SEQ ID NO.7); The amino acid sequence of the third heavy chain variable region (T508-A1T-ACTA2-VH3) is: EVKLQQSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPSNDYAKYDPKFQGKATITSDTSSNTAYLQVSSLTSEDTAVYYCGLYYGRSIDYWGQGTTLTVSS(SEQ ID NO.8); The amino acid sequence of the fourth heavy chain variable region (T508-A1T-ACTA2-VH4) is: QVQLQESGTELASPGASVKLSCKASGYIFSDYWMQWVKERPGQGLEWIGTIYPGDGDARYNLKFKNKAALTADKSSNTAYMLLNNLASEDSAVYYCARYWDYDVGGFTYWGQGSLVTVSA (SEQ ID NO. 9).

[0050] Example 3: Expression screening of recombinant monoclonal ACTA2 1. Expi CHO-S cell eukaryotic expression test The light chain and heavy chain pairings of four recombinant monoclonal antibodies were VK1 / VH1 (R1), VK1 / VH2 (R2), VK1 / VH3 (R3), and VK1 / VH4 (R4), and the ExpiCHO-S™ Expression System was used for 2 mL eukaryotic cell transfection, antibody expression, and purification.

[0051] The specific transfection method based on ExpiCHO-S™ Expression System is as follows: Select a density of 3×10 6 ~1×10 7 Expi CHO-S live cells / mL and the seed cells with cell viability greater than 95% were transfected; the reagents required for transfection were calculated according to the volume of the cell suspension, so that the final cell density in each 2 mL transfection system was 3×10 6 viable cells / mL, incubated at 37°C in a shaking incubator with appropriate humidity, 8% CO2, and a rotation speed of 125 rpm for 30 min; the transfection reagent was PEI, and the diluent was a NaCl solution containing 5% HEPES. The final cell density was 2×10 6 viable cells / mL, total volume 2 mL, plate six-well plates, set up a positive control; use 100 µL NaCl solution to dilute 4 µg plasmid DNA, use 100 µL NaCl solution to dilute 10 µg PEI solution, use a pipette to add the diluted plasmid dropwise to each well, set up a positive control, put it back on the shaker, incubate for 5 min, add the PEI dilution dropwise to each well, gently shake the six-well plate, return to the shaker for culture, 37℃ shaking incubator, appropriate humidity, 8% CO2, speed of 125 rpm; 24 hours after transfection, add 50 µL sodium butyrate solution and 100 µL EN3 solution to each well, and start sampling from the culture medium to detect recombinant protein expression 72 hours later.

[0052] Purification was performed using pure beads, and the specific steps are as follows: Take 100 µL of pure beads (Protein A, a membrane protein) to a 1.5 mL EP tube, wash twice with 1 mL PBS buffer, and add 400 µL PBS buffer to prepare 20% beads; take the eukaryotic transfected cells, centrifuge at 1000 rpm for 5 minutes, take 40 µL of supernatant sample, take an appropriate amount of precipitate sample, and dilute it with 40 µL PBS buffer; collect the supernatant, add 50 µL of 20% beads, and incubate at room temperature for 30 min; collect the incubated beads, gradually transfer the incubated supernatant to a new 1.5 mL EP tube, centrifuge, take 40 µL of supernatant as the flow-through sample, discard the rest of the flow-through, repeat this step to collect all beads; add 10 µL 5×Loading Buffer to the supernatant, precipitate, and flow-through, and add 20 µL 5×Loading Buffer to the beads; incubate all samples at 100°C for 10 Boil for min and perform SDS-PAGE gel electrophoresis.

[0053] The results are as follows Figure 8As shown, after using the ExpiCHO-S™ Expression System for 2 mL eukaryotic cell transfection and antibody expression and purification, the reducing SDS-PAGE electrophoresis analysis showed clear bands at 25 kDa and 55 kDa with no other bands, which was consistent with the expected target bands.

[0054] 2. Preparation of ACTA2 protein The primer sequences of ACTA2 were designed through UNIPROT (https: / / www.uniprot.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) websites as follows: ACTA2 upstream primer: GGTGGACAGCAAATGGGTTGTGAAGAAGAGGAC (SEQ ID NO.32), ACTA2 downstream primer: GTGGTGGTGGTGGTGGTGGAAGCATTTGCGGTGG (SEQ ID NO.33); the primers were synthesized by Qingke Bio. The target fragment of ACTA2 was amplified by polymerase chain reaction (as shown in Tables 3 and 4). After amplification, 1 μL of the recombinant product of ACTA2 was taken for agarose gel electrophoresis verification, and the target fragment of ACTA2 was recovered using Tiangen Ordinary Agarose Gel DNA Recovery Kit (Cat. No.: DP209). The expression vector pET21b was digested with restriction endonucleases BamH I and XhoI, and the target fragment of ACTA2 and the linearized vector were then recombined and transformed into competent cells BL21 (stored in this laboratory). Single white colonies were picked for sequence identification. The identification results were as follows: Figure 7 As shown in Figure A (ACTA2 colony PCR agarose gel electrophoresis).

[0055] Take the bacterial liquid of the correctly identified cloned strain and expand the culture in LB medium (Luria-Bertani medium) containing ampicillin, and then induce protein expression under the conditions of 0.5 mmol / L IPTG (isopropyl-β-D-thiogalactoside) and 37°C. Collect the bacteria, use an ultrasonic disruptor to disrupt the bacteria and collect the supernatant. Add the supernatant to the nickel affinity filler at a certain flow rate, and then elute the protein with 20 mmol / L, 50 mmol / L, 100 mmol / L, 250 mmol / L and 500 mmol / L imidazole elution buffers in sequence. Take 20 µL of the flow-through and different concentrations of imidazole elution buffers for denaturing reduced polyacrylamide gel electrophoresis. Concentrate the protein eluted with 50 mmol / L imidazole elution buffer and perform non-denaturing non-reducing polyacrylamide gel electrophoresis. The electrophoresis results are as follows. Figure 7 As shown in Figure B (ACTA2 polyacrylamide gel electrophoresis).

[0056] 3. Mouse anti-human ACTA2 recombinant monoclonal antibody ELISA test titer In order to further screen the optimal mouse anti-human ACTA2 recombinant monoclonal antibody sequence, the ExpiCHO-S™ Expression System was used for 2 mL eukaryotic cell transfection, antibody expression and purification, and the titer was tested by supernatant ELISA.

[0057] The specific titer determination method is as follows: 1) Coating: Prepare carbonate buffer solution, dilute ACTA2 protein to 2 µg / mL, add 100 µL / well of diluted ACTA2 protein to a 96-well ELISA plate, and react overnight at 4°C. 2) Washing: Pour out the liquid in the 96-well ELISA plate, wash the plate three times with PBST buffer, and then pat the liquid in the 96-well ELISA plate dry on filter paper; 3) Blocking: Add 200 µL of 2% BSA (bovine serum albumin) to each well and incubate at 37°C for 2 hours; 4) Washing: Pour out the liquid in the 96-well ELISA plate, wash the plate three times with PBST buffer, and then pat the liquid in the 96-well ELISA plate dry on filter paper; 5) Adding primary antibody: setting the antibody concentrations to 10 ng / mL, 3.33 ng / mL, and 1.11 ng / mL, diluting the antibody according to these three gradient concentrations, adding 100 µL of the diluted antibody (mouse anti-human ACTA2 recombinant monoclonal antibody prepared by the present invention) to each well, placing in an incubator, and incubating at 37°C for 1 hour; 6) Washing: Pour out the liquid in the 96-well ELISA plate, wash the plate three times with PBST buffer, and then pat the liquid in the 96-well ELISA plate dry on filter paper; 7) Add enzyme-labeled secondary antibody: add 100 µL of 1:4000 enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) to each well and incubate at 37°C for 40 min; 8) Washing the plate: Pour out the liquid in the 96-well ELISA plate, wash the plate three times with PBST buffer, and then pat the liquid in the 96-well ELISA plate dry on filter paper; 9) Color development: Add 100 µL of color development solution to each well, place incubator, incubate at 37°C for 10 min, and add 50 µL of 2 mol / L sulfuric acid solution to terminate the reaction; 10) Determine OD value: Use a microplate reader to measure the absorbance (OD 450) value of each well.

[0058] The results showed that the recombinant antibodies paired with T508-A1T-ACTA2-VK1 / VH2 and T508-A1T-ACTA2-VK1 / VH3 were expressed as Figure 8 As shown, A is the expression of T508-A1T-ACTA2-VH2 / VK1 and T508-A1T-ACTA2-VH1 / VK1, band 4 is T508-A1T-ACTA2-VH2 / VK1, and band 8 is T508-A1T-ACTA2-VH1 / VK1; B is the expression of T508-A1T-ACTA2-VH3 / VK1 and T508-A1T-ACTA2-VH4 / VK1, band 8 is T508-A1T-ACTA2-VH3 / VK1, and band 12 is T508-A1T-ACTA2-VH12 / VK1; T508-A1T-ACTA2-VK1 / VH3 ELISA results showed weak positive, while T508-A1T-VK1 / VH2 The ELISA results showed positive, strong signals, and good recognition of the coated ACTA2 protein. Fig.11 As shown. Figure A shows the titer results of T508-A1T-ACTA2-VH1 / VK1(R1), Figure B shows the titer results of T508-A1T-ACTA2-VH2 / VK1(R2), Figure C shows the titer results of T508-A1T-ACTA2-VH3 / VK1(R3), and Figure D shows the titer results of T508-A1T-ACTA2-VH4 / VK1(R4); The T508-A1T-VK1 / VH2 bacterial solution was sent to a sequencing company for sequencing analysis. The sequencing results are as follows Fig. 9 As shown, the results show that the sequencing peak graph presents obvious, clear and symmetrical peaks, with good resolution and signal-to-noise ratio, and no abnormal peaks or mixed peaks, indicating that the sequencing results are reliable and can be used for subsequent experiments.

[0059] In order to further obtain milligram-level ACTA2 recombinant antibodies for sample verification and characterization, this study used the ExpiCHO-S™ Expression System to perform 60 mL eukaryotic pilot expression and antibody affinity chromatography purification of the screened T508-A1T-ACTA2-VK1 / VH2. The purified recombinant antibodies were analyzed by reducing SDS-PAGE (e.g. Fig.10 As shown in the figure, there are clear bands at 25 kDa and 55 kDa without any other bands, which is consistent with the expected target bands.

[0060] Embodiment 4: In this example, the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the method described in Examples 1-3 was subjected to ELISA affinity detection, and the detection process was as follows: To further characterize the affinity of ACTA2 recombinant antibody, indirect ELISA method was used for identification, and then OD 450 nm is the ordinate, and the logarithm of the antibody concentration is the abscissa. The two curves were fitted using the software Origin 8.5 to calculate the OD max / 2 corresponding to the antibody concentration (mol / L), calculate the antibody affinity constant Ka. The result is as follows Fig.12 As shown, A is the concentration of the coated recombinant protein 0.5 μg / mL, and B is the concentration of the coated recombinant protein 1 μg / mL; Calculated according to the affinity constant Ka (Formula 1-1): Ka = (n-1) / 2( n[Ab]1-[Ab]2) (Formula 1-1) In the formula, [Ab]1 and [Ab]2 are two different coating concentrations at OD max / 2 corresponds to the antibody concentration (mol / L) [Ab]1 corresponds to a relatively low coating concentration, [Ab]2 corresponds to a relatively high coating concentration, and n is the ratio of the two groups of coating concentrations (n>1). The affinity of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 was calculated. The results showed that the affinity of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 was 6.6×10 10 L / mol, affinity greater than 10 7 L / mol is a high-affinity antibody, indicating that the variable region encoding gene of the anti-ATCA2 recombinant monoclonal antibody prepared by the present invention has a high affinity.

[0061] Embodiment 5: In this example, the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the method described in Examples 1-3 was subjected to Western Blot detection. The detection process is as follows: In order to verify the binding ability and specificity of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared by the present invention to the natural ACTA2 antigen in the cell sample, in this example, the mouse anti-human ACTA2 recombinant monoclonal antibody and the commercial antibody (Leica) were subjected to Western Blot detection, and it was detected that Human colon, Human spleen, Rar brain, Rat heart, Mouse heart, Mouse brain, 293T, A431, MCF-7, HepG2, K-562 (stored in this laboratory) had bands at the corresponding molecular weight positions. The results showed that the ACTA2 protein in the positive cells had specific recognition ability, and there was no target band in Human colon, and the result showed negative.

[0062] The results are as follows Fig.13 As shown. The results showed that there was no significant statistical difference in the detection of ACTA2 protein levels in the above cells (p>0.05). This shows that the ACTA2 recombinant antibody prepared in this study can bind to the common epitope of mutant and non-mutant ACTA2 proteins and has good specificity and sensitivity in Western Blot experiments. Embodiment 6: In this example, the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the method described in Examples 1-3 was subjected to immunohistochemical detection, and the detection process was as follows: In order to further verify the recognition ability of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared by the present invention on the expression of ACTA2 protein in pathological tissue samples, this example performed immunohistochemical verification on more than 40 tissue samples, and the specific steps are as follows: (1) Preparation of tissue wax blocks. Seven types of human cancer tissue wax blocks were selected, including rectal adenocarcinoma, liver cancer, breast cancer, well-differentiated squamous cell carcinoma, breast phyllodes carcinoma, renal carcinoma, and sigmoid colon adenocarcinoma, as well as three types of animal tissue wax blocks, including mouse liver cancer and normal monkey tissue (testis, duodenum). Four consecutive sections were taken for each tissue. (2) Detection was performed using commercially available ACTA2 antibody and self-developed ATCTA2 recombinant antibody; (3) Place the paraffin sections in a 60°C oven for 60 min, and then use a Leica multifunctional stainer for dewaxing and hydration; (4) Perform EDTA high-temperature antigen repair on the antigen: Take a certain amount of EDTA antigen repair working solution with pH 9.0 in the repair box. The amount of repair solution must be enough to immerse the entire slice. Put the repair box into a pressure cooker with an appropriate amount of tap water, heat it to boiling over high heat, place the dewaxed and hydrated tissue slices on a high-temperature staining rack, and then slowly place the staining rack into the repair box. Cover the lid without buckling the pressure valve. After 20 minutes, disconnect the power supply of the pressure cooker, remove the valve and open the lid, take out the repair box, leave it at room temperature for 20 minutes to cool naturally, and then take out the slice; (5) Rinse with pure water twice, 5 min each time, add 3% H2O2, and incubate at room temperature in a wet box for 20 min; (6) Rinse with PBS buffer three times, 5 min each time. Add sheep serum to block the cells and incubate in a wet box at 37°C for 30 min. (7) Discard the blocking solution and add primary antibodies diluted 1:100 and 1:200. Incubate at 4°C overnight and then rewarm at 37°C for 30 min. (8) Discard the primary antibody and soak in PBS buffer three times, 5 min each time; (9) Add goat anti-mouse secondary antibody and incubate in a wet box at 37°C for 30 min. Discard the secondary antibody. (10) Soak in PBS buffer for 3 times, 5 min each time, add DAB working solution, observe the color development under a microscope and control the time. After the color development is complete, rinse with pure water; (11) Restain with hematoxylin for 3 seconds, rinse with tap water for 5 minutes, soak in 1% hydrochloric acid ethanol for 1 second, and soak in tap water for 2 minutes; (12) Stain with Scott's stain for 2 min, soak in tap water for 2 min, and examine under a microscope; (13) Use Leica multifunctional staining machine to dehydrate, transparentize, and seal the slides, and finally take photos and scan the slides.

[0063] Some of the results are as follows Fig.14 As shown. Fig.14 A is the immunohistochemical detection of human breast cancer / human rectum by ACTA2 recombinant antibody, B is the IF result of immunohistochemical detection of subcellular localization in human liver by ACTA2 recombinant antibody, C is the IF result of immunohistochemical detection of subcellular localization in human smooth muscle by ACTA2 recombinant antibody, D is the A549 confocal and 64x image of immunohistochemical detection of cellular and subcellular localization by ACTA2 recombinant antibody.

[0064] The results showed that ACTA2 was selectively expressed in smooth muscle cells and myoepithelial cells, and was mainly localized to actin filaments. The antibody can specifically bind to the target antigen, which is consistent with the expected tissue localization, tissue sublocalization, cellular localization and subcellular localization; and the signal is strong, and it is expected to be well applied in IHC. ACTA2 is mainly localized to actin filaments, and the results showed that the antibody can specifically bind to the target antigen, which is consistent with the expected tissue localization, tissue sublocalization, cellular localization and subcellular localization; and the signal is strong, and it is expected to be well applied in IF. ACTA2 is mainly localized to actin filaments, and the results showed that the antibody can specifically bind to the target antigen, which is consistent with the expected tissue localization, tissue sublocalization, cellular localization and subcellular localization; and the signal is strong, and it is expected to be well applied in ICC.

[0065] In summary, the present invention successfully prepared a mouse anti-human ACTA2 recombinant monoclonal antibody, and the antibody has high titer and affinity. The Western Blot and immunohistochemistry detection results of the ACTA2 recombinant antibody are similar to those of the commercial ACTA2 antibody, and in some tissue samples, the immunohistochemistry positive rate is significantly higher than that of the commercial ACTA2 antibody. The antibody can be used for Western Blot and immunohistochemistry detection, and can provide preliminary data support for clinical pathological diagnosis.

[0066] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A gene encoding the variable region of a recombinant monoclonal antibody against ATCA2, characterized in that: The anti-ATCA2 recombinant monoclonal antibody variable region encoding gene includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes one of the amino acid sequences shown in SEQ ID NO.6-9; The light chain variable region includes the amino acid sequence shown in SEQ ID NO.

10.

2. A nucleic acid, characterized in that The nucleic acid encodes the heavy chain variable region and the light chain variable region of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 as claimed in claim 1.

3. A nucleic acid according to claim 2, characterized in that The nucleic acid comprises one of the nucleotide sequences shown in SEQ ID NO.1-5.

4. A nucleic acid according to claim 3, characterized in that The nucleotide sequence shown in SEQ ID NO.1 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.6 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.2 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.7 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.3 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.8 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.4 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.9 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2; The nucleotide sequence shown in SEQ ID NO.5 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.10 in the variable region encoding gene of the recombinant monoclonal antibody against ATCA2.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to any one of claims 2 to 4.

6. A recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector includes pcDNA3.4 plasmid.

7. A recombinant expression cell, characterized in that: The recombinant expression cell contains the nucleic acid according to any one of claims 2 to 4, or the recombinant expression vector according to claim 5 or 6.

8. A method for preparing a gene encoding a variable region of a recombinant monoclonal antibody against ATCA2, characterized in that: The preparation method comprises: using the recombinant expression vector as claimed in claim 5 or 6 to transfect cells to obtain recombinant expression cells, culturing the recombinant expression cells obtained after transfection, collecting and purifying the supernatant, and obtaining the variable region encoding gene of the recombinant monoclonal antibody against ACTA2.

9. Use of the nucleic acid according to any one of claims 2 to 4, or the recombinant expression vector according to claim 5 or 6, or the recombinant expression cell according to claim 7 in preparing a gene encoding a variable region of a recombinant monoclonal antibody against ACTA2.

10. Use of the recombinant monoclonal antibody variable region encoding gene against ACTA2 as claimed in claim 1, or the nucleic acid as claimed in any one of claims 2 to 4, or the recombinant expression vector as claimed in claim 5 or 6, or the recombinant expression cell as claimed in claim 7, or the preparation method as claimed in claim 8 in detecting ACTA2 protein molecules or preparing a device for detecting ACTA2 protein molecules.

Images