Recombinant monoclonal antibody variable region encoding gene against atca2 and preparation method

Abstract

The application belongs to the technical field of biology and particularly relates to a recombinant monoclonal antibody variable region coding gene against ATCA2 and a preparation method. The recombinant monoclonal antibody variable region coding 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. The application recombines a mouse anti-human ACTA2 recombinant monoclonal antibody by using a hybridoma technology, the recombinant monoclonal antibody has a clear structure, a high titer and better stability, can effectively solve the problems existing in traditional antibody preparation, can be applied to the detection and screening fields such as immunohistochemistry, indirect ELISA, antibody chip preparation and immunofluorescence, and is favorable for obtaining accurate evaluation and detection results.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a variable region encoding gene of a recombinant monoclonal antibody against ATCA2 and its preparation method. Background Technology

[0002] Antibodies are protective proteins produced by the body in response to antigen stimulation; they are a class of immunoglobulins that specifically bind to antigens. ACTA2 (Actin, aortic smooth muscle) is a G protein-coupled receptor (GPCR), a dimer composed of an α subunit and a β subunit. ACTA2 plays a crucial role in controlling cardiovascular activity and maintaining blood pressure. In the heart, ACTA2 is located in the intracellular region of the cardiac β1 receptor and acts as a ligand-binding receptor for the cardiac β1 receptor. The cardiac β1 receptor is a GPCR located on the cell 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. Therefore, ACTA2 may be a promising prognostic biomarker and anticancer drug target for metastatic lung adenocarcinoma.

[0003] Traditional antibody preparation typically involves immunizing animals to obtain monoclonal and polyclonal antibodies. Monoclonal antibodies are produced by immunizing host animals with a target protein, then extracting B cells that recognize and respond to that antigen, fusing them with myeloma cells to form hybridoma cells, thus creating antibodies. Polyclonal antibodies are prepared by collecting blood from animals immunized with a target antigen. However, currently prepared antibodies have several problems, such as low antibody titers; the purchased antibody for detecting protein X preferentially binds to protein Y, or even not binds to X at all; poor antibody stability; large batch-to-batch variability; and poor reproducibility, resulting in unrepeatable experimental results when repeating previous experiments with different batches of antibodies.

[0004] The aforementioned problems have led to erroneous scientific discoveries and wasted research efforts, resulting in significant losses in fields such as cancer, metabolism, aging, immunology, cell signal transduction, and any complex biomolecular research. Therefore, developing a method for preparing anti-ACTA2 antibodies with a well-defined structure, high potency, and high stability is of great importance. Summary of the Invention

[0005] To address the above-mentioned deficiencies or improvement needs of existing technologies, this invention provides a variable region encoding gene of a recombinant monoclonal antibody against ATCA2, a preparation method, and its application. By utilizing hybridoma technology to prepare a mouse anti-human ACTA2 recombinant monoclonal antibody, the recombinant antibody has a well-defined structure, high potency, and better stability.

[0006] The first objective of this invention is to provide a variable region encoding gene of a recombinant monoclonal antibody against ATCA2.

[0007] A second aspect of the present invention is to provide a nucleic acid.

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

[0009] The fourth aspect of this invention is to provide a recombinant expression cell.

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

[0011] The sixth aspect of this invention aims to provide the use of the nucleic acid described in the second aspect, or the variable region coding gene of the recombinant monoclonal antibody 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 the variable region coding gene of the recombinant monoclonal antibody against ATCA2.

[0012] The seventh aspect of this invention aims to provide the 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 the detection of ACTA2 protein molecules, or in the preparation of an apparatus for detecting ACTA2 protein molecules.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a variable region encoding gene for a recombinant monoclonal antibody against ATCA2, the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising any one of the amino acid sequences shown in SEQ ID NO. 6-9; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 10.

[0015] Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2 includes the amino acid sequence shown in SEQ ID NO. 6 in the heavy chain variable region and the amino acid sequence shown in SEQ ID NO. 10 in the light chain variable region;

[0016] Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2 includes the amino acid sequence shown in SEQ ID NO.7 in the heavy chain variable region and the amino acid sequence shown in SEQ ID NO.10 in the light chain variable region;

[0017] Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2 includes the amino acid sequence shown in SEQ ID NO.8 in the heavy chain variable region and the amino acid sequence shown in SEQ ID NO.10 in the light chain variable region;

[0018] Furthermore, the variable region encoding gene of the above-mentioned recombinant monoclonal antibody against ATCA2 includes the amino acid sequence shown in SEQ ID NO.9 in the heavy chain variable region and the amino acid sequence shown in SEQ ID NO.10 in the light chain variable region.

[0019] In a second aspect, the present invention provides a nucleic acid that encodes the heavy chain variable region and the light chain variable region of the variable region encoding gene of the aforementioned recombinant monoclonal antibody against ATCA2.

[0020] Furthermore, the nucleic acid encoding the variable region of the recombinant monoclonal antibody against ATCA2 includes:

[0021] 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;

[0022] 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 coding gene of the recombinant monoclonal antibody against ATCA2;

[0023] 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 coding gene of the recombinant monoclonal antibody against ATCA2;

[0024] 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 coding gene of the recombinant monoclonal antibody against ATCA2;

[0025] 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 coding gene of the recombinant monoclonal antibody against ATCA2.

[0026] A third aspect of the present invention provides a recombinant expression vector containing the aforementioned nucleic acid.

[0027] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the aforementioned nucleic acid.

[0028] In a fourth aspect, the present invention provides a recombinant expression cell containing the nucleic acid encoding the variable region of the recombinant monoclonal antibody against ATCA2, or the recombinant expression vector described above.

[0029] In a fifth aspect, the present invention provides a method for preparing the variable region coding gene of a recombinant monoclonal antibody against ATCA2. The method comprises: transfecting cells with the above-mentioned recombinant expression vector to obtain recombinant expression cells, culturing and transfecting the cells to obtain recombinant expression cells, collecting the supernatant and purifying it to obtain the variable region coding gene of a recombinant monoclonal antibody against ATCA2.

[0030] A sixth aspect of the present invention provides the use of the above-described nucleic acid, or the above-described recombinant monoclonal antibody variable region coding gene against ATCA2, or the above-described recombinant expression vector, or the above-described recombinant expression cell in the preparation of the recombinant monoclonal antibody variable region coding gene against ATCA2.

[0031] A seventh aspect of the present invention provides the application of the above-described recombinant monoclonal antibody against ATCA2, the variable region encoding gene, or the above-described nucleic acid, or the above-described recombinant expression vector, or the above-described recombinant expression cell, or the above-described preparation method in detecting ACTA2 protein molecules, or in preparing an apparatus for detecting ACTA2 protein molecules.

[0032] Furthermore, the detection of ACTA2 protein molecules includes immunohistochemistry and affinity assay methods.

[0033] Furthermore, the immunohistochemical assay includes: dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting, and microscopic examination.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention provides a variable region coding gene for a recombinant monoclonal antibody against ATCA2, its preparation method, and its applications. By utilizing hybridoma technology to recombinant monoclonal antibodies, a variable region coding gene for a recombinant monoclonal antibody against ATCA2 is prepared, resulting in an antibody with a well-defined structure, high titer, and good stability. This antibody exhibits high specificity and sensitivity in binding to the ACTA2 protein molecule, specifically recognizing and detecting ACTA2 protein expression in tumor tissues. It shows high positive expression when detecting ACTA2 protein. Therefore, this antibody can be applied in immunohistochemistry, indirect ELISA, antibody chip preparation, immunofluorescence, and other detection and screening fields, facilitating accurate assessment and detection results.

[0036] The present invention provides a recombinant monoclonal antibody against ATCA2, including its variable region encoding gene, preparation method, and application. It has the characteristics of high specificity and strong positive signal, and can provide a reference for clinical prognostic assessment. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart illustrating the preparation and expression process of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of this invention.

[0039] Figure 2 This is a gel electrophoresis image of total RNA from hybridoma cells in this invention;

[0040] Figure 3 This is a diagram showing the extraction of the variable region encoding gene of the recombinant monoclonal antibody against ACTA2 in this invention.

[0041] Figure 4 This is a diagram of the amplification of the mouse anti-human ACTA2 full-length recombinant antibody gene in this invention;

[0042] Figure 5 This is a diagram of the mouse heavy chain constant region vector pFUSEss-CHIg-mG2B of the present invention;

[0043] Figure 6 This is a diagram of the mouse light chain constant region vector pFUSE2ss-CLIg-mk of the present invention;

[0044] Figure 7 This is the result of the preparation of ACTA2 protein in this invention;

[0045] Figure 8This is an image showing the SDS-PAGE detection results of the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of this invention;

[0046] Figure 9 This is a sequencing result of the T508-A1T-VK1 / VH2 gene sequence encoding the variable region of the optimal anti-ATCA2 recombinant monoclonal antibody of this invention.

[0047] Figure 10 The image shows the results of SDS-PAGE analysis of the variable region encoding gene sequence T508-A1T-VK1 / VH2 of the optimal anti-ATCA2 recombinant monoclonal antibody of this invention using the ExpiCHO-S™ Expression System.

[0048] Figure 11 This is a graph showing the titer detection results of the ACTA2 recombinant antibody of this invention;

[0049] Figure 12 This is the affinity constant curve of the ACTA2 recombinant antibody of the present invention;

[0050] Figure 13 This is a Western Blot diagram of the variable region coding gene of the recombinant monoclonal antibody against ATCA2 of this invention.

[0051] Figure 14 This is a diagram showing the immunohistochemical verification results of tissue samples containing the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 of this invention. Detailed Implementation

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0053] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0054] The flowchart of the method for preparing the variable region encoding gene of a recombinant monoclonal antibody against ATCA2 according to the present invention is shown below. Figure 1 As shown.

[0055] The following specific examples will provide further details.

[0056] This invention provides a method for preparing a mouse anti-human ACTA2 recombinant monoclonal antibody, specifically including the steps shown in Examples 1-3.

[0057] Example 1: Gene fishing for the variable region of mouse monoclonal antibody

[0058] 1. Results of RNA extraction and reverse transcription from hybridoma cells

[0059] Hybridoma cells were obtained by fusing mouse spleen cells with SP2 / 0 (mouse myeloma cells); total RNA was extracted from hybridoma cells using the Trizol method, and clear 28S and 18S bands were observed by agarose gel electrophoresis (e.g., ...). Figure 2 As shown in the figure, the RNA integrity is good. The RNA concentration and purity were measured, and the result was D(260 nm) / D(280 nm) = 1.98, which meets the requirements of this experiment.

[0060] 3) Using the total RNA prepared above as a template, cDNA was synthesized by reverse transcription using the Tiangen FastKing One-Step RT-PCR Kit (catalog number: KR123) reverse transcription kit. The amplification system and program are shown in Tables 1 and 2. The amplification system included 4 µL of 5×FastKing-RT SuperMix; 50 ng - 2 µg of total RNA; and RNase-free ddH2O to a final volume of 20 µL. The amplification reaction program included incubation at 42℃ for 15 min, followed by incubation at 95℃ for 3 min.

[0061] Table 1 RNA reverse transcription system

[0062]

[0063] Table 2 shows the program settings as follows:

[0064]

[0065] 2. PCR amplification of the variable region of mouse monoclonal antibody

[0066] Using the cDNA prepared in step 1 as a template, the complete VH gene was amplified by mixing one mouse VH upstream degenerate primer and one VH downstream degenerate primer. The complete VK gene was amplified by mixing two mouse VK upstream degenerate primers and two VK downstream degenerate primers in a specific ratio. The amplification system, program, and primer sequences are shown in Tables 3, 4, and 5. The amplification system included 25 µL of buffer; 1 µL of Phanta Max Super-Fidelity DNA Polymerase; 20 µL of ddH2O; 1 µL of upstream degenerate primer mixture; 1 µL of downstream degenerate primer mixture; and 1 µL of template cDNA. The amplification reaction program included: incubation at 95°C for 3 min, followed by 30 cycles of incubation at 95°C, 56°C, and 72°C for 30 s each, and a final incubation at 72°C for 5 min.

[0067] The agarose gel electrophoresis results of the amplification products are as follows: Figure 3As shown in Figure A, the PCR amplification of the heavy / light chain variable region genes is as follows: 1-6: heavy chain variable region amplification, 7-12: light chain variable region amplification. The VH gene fragment is approximately 350-400 bp in length, and the VK gene fragment is approximately 350 bp in length, which is consistent with the length of the target fragment.

[0068] Table 3 PCR amplification system

[0069]

[0070] Table 4 PCR Amplification Program

[0071]

[0072] Table 5 Primer Sequences

[0073]

[0074] 3. Purify the V region gene and ligate it with the T-vector.

[0075] The PCR product from step 2 was purified using the Tiangen agarose gel DNA recovery kit (catalog number: DP209). The VH and VK genes were ligated into the pGEM-T vector, respectively, and transformed into *E. coli*. The ligation 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.

[0076] The specific method is as follows:

[0077] (1) Pick a single colony from the petri dish that has been cultured at 37°C for 16-20 h, inoculate it into 5 mL of LB medium, place it in a shaker at 37°C, set the rotation speed to 220 rpm / min, and culture for 6-8 h;

[0078] (2) The above initial culture was inoculated 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 incubated overnight at 18°C ​​and 180 rpm on a shaker.

[0079] (3) The next morning, the OD 600 values ​​of the three culture flasks were measured every 45 minutes. When the OD 600 of the culture was 0.55, the culture flasks were placed on ice for 10 minutes.

[0080] (4) Dispense 200 mL of culture into four 50 mL centrifuge tubes, centrifuge at 5000 rpm for 10 min at 4℃, and collect the bacterial cells;

[0081] (5) Discard the culture medium, blot the remaining liquid with absorbent paper, and then resuspend the bacterial pellet in 20 mL of pre-cooled Inoue transformation buffer in each tube. (Note: Resuspend gently; do not mix with a shaker or pipetting.)

[0082] (6) Place it in a centrifuge, adjust the temperature to 4℃, adjust the speed to 5000 rpm, centrifuge for 10 min and collect the bacterial cells;

[0083] (7) Discard the culture medium, absorb the remaining liquid with absorbent paper, combine the 4 tubes and add 20 mL of pre-cooled Inoue conversion buffer to gently resuspend the precipitate;

[0084] (8) Add 1.5 mL of DMSO solution, gently mix the bacterial suspension, place it on ice for 10 min, quickly dispense the suspension into cooled sterile microcentrifuge tubes, seal the tube openings, immerse the competent cells in liquid nitrogen to freeze them quickly, and store them in a -80℃ freezer for later use.

[0085] (9) Pick a single white colony with a sterile 10 µL pipette tip and place it into a new 1.5 mL EP tube. Add 1 mL of liquid LB medium containing ampicillin antibiotic to the tube and place it in a double-layer constant temperature shaker. Incubate at 220 rpm and 37℃ for 3-6 h. Remove the bacterial solution for later use.

[0086] (10) Prepare the reaction system according to Tables 6.1 and 6.2 below and mix well, then place it in a PCR instrument;

[0087] Table 6.1 Connection Reaction System

[0088]

[0089] Table 6.2 Bacterial PCR Amplification System

[0090]

[0091] (11) The bacterial culture PCR amplification program settings are shown in Table 6.3:

[0092] Table 6.3 Bacterial PCR Amplification Program

[0093]

[0094] (12) Three light chain and three heavy chain variable region genes from each of the clones that were positive for bacterial culture PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Colony PCR was performed using universal primers CMV-F. The target fragment size was about 500 bp. Eighteen T clones were selected for the heavy chain, of which strains 3, 5, 8, 10, 13, 14, and 18 were correct (P). T 21 T clones were selected for the light chain, of which strains 1, 3, 5, 8, 10, 13, 15, 18, 19, 20, and 21 (e.g., ...) were correct (P). Figure 3 As shown in Figure B, which shows the identification of T-clone colonies in the heavy chain variable region (lanes 1-18 represent 1-18 T-clones in the heavy chain variable region, with colonies 3, 5, 8, 10, 13, 14, and 18 being correct P-clones); and Figure C, which shows the identification of T-clone colonies in the light chain variable region (lanes 1-21 represent 1-21 T-clones in the light chain variable region, with colonies 1, 3, 5, 8, 10, 13, 15, 18, 19, 20, and 21 being correct P-clones). Seven positive heavy chain clones and eleven positive light chain clones with bright bands were sent to Kexin Technology for sequencing.

[0095] 4. Amplification of the full-length recombinant antibody gene

[0096] Seven heavy chain T clones and eleven light chain T clones were analyzed using IMGT or QUEST online analysis software. One effective light chain and four effective heavy chains were identified and named T508-A1T-ACTA2-VK1, T508-A1T-ACTA2-VH1, T508-A1T-ACTA2-VH2, T508-A1T-ACTA2-VH3, and T508-A1T-ACTA2-VH4, respectively. The results of one effective heavy chain (T508-A1T-ACTA2-VH4) and one light chain (T508-A1T-ACTA2-VK1) are used as representative analyses. The T508-A1T-ACTA2-VK1 gene was analyzed for its effectiveness and VDJ gene family. The full-length variable region of this functional light chain is 334 bases. The functional light chain belongs to Musmus IGKV3-10. The 01F family showed a 96.22% matching rate in region V and 92.11% in region J. Analysis of the T508-A1T-ACTA2-VH2 heavy chain gene for efficacy and the VDJ gene family revealed that the full-length variable region of this functional heavy chain is 352 bases. All functional heavy chains belong to Musmus IGHV14-3. For the 02 F family, the matching rate for region V was 96.18%, and for region J it 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 for 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.

[0097] Table 7. Light Chain Structural Domain Division of T508-A1T-ACTA2-VK1

[0098]

[0099] Table 8. Heavy chain domain division of T508-A1T-ACTA2-VH2

[0100]

[0101] Table 9. Light chain variable region structures and their coding genes

[0102]

[0103] Table 10 Heavy chain variable region structures and their coding genes

[0104]

[0105] Example 2: Construction of a full-length recombinant antibody vector against human anti-ACTA2 in mice

[0106] To construct a full-length recombinant antibody plasmid against human ACTA2 in mice, 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) selected in Example 1 were constructed into the pFUSE2ss-CLIg-mk and pFUSEss-CHIg-mG2B vectors, respectively. PCR amplification primers were designed based on the upstream and downstream restriction enzyme sites of the vectors, and the full-length recombinant antibody gene was amplified. The results are as follows: Figure 4 As shown in Figure A, bands 1-6 represent the amplification of the heavy chain variable region, and bands 7-12 represent the amplification of the light chain variable region. 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 size is consistent with the expected theoretical value.

[0107] Subsequently, to determine whether the heavy 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, colony identification was performed using the hEF1-HTLV prom and SV40 universal primers for four T clones of the heavy chain variable region and five T clones of the light chain variable region. The results are as follows: Figure 4 As shown in Figure B (Figure B shows the identification of colonies of heavy chain variable region gene clones in 1-4 and light chain variable region gene clones in 5-9), 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.

[0108] The specific method is as follows:

[0109] 1. PCR amplification primers were designed based on the upstream and downstream restriction sites of the vector, as shown in Table 11. Then, the heavy and light chain variable region genes that were confirmed to be correct and functional were used as templates by sequencing.

[0110] Table 11 PCR amplification primer sequences

[0111]

[0112] 2. Using mouse heavy chain constant region vector pFUSEss-CHIg-mG2B ( Figure 5 ) and mouse light chain constant region vector pFUSE2ss-CLIg-mk ( Figure 6 Using the above mouse antibody heavy chain and light chain constant region amplification products as the backbone, the amplification products were gel-recovered and ligated into the mouse heavy chain constant region vector pFUSEss-CHIg-mG2B and the mouse light chain constant region vector pFUSE2ss-CLIg-mk, respectively.

[0113] (1) Light chain constant region modification: The vector pFUSE2ss-CLIg-mk was 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, digested with EcoRI and XhoI and ligated into 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).

[0114] 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).

[0115] (2) Modification of heavy chain constant region: The vector pFUSEss-CHIg-mG2B was digested with NheI and EcoRI; the mouse heavy chain IgG2B constant region gene was amplified, and the target band was inserted after double digestion with NheI and EcoRI and sequenced for identification.

[0116] The nucleotide sequence encoding the first heavy chain variable region (T508-A1T-ACTA2-VH1) is shown below: GAGGTCAAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAGGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGGACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCGTTATACCCACGGGCTACTTTGACTACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCAG (SEQ ID NO.1);

[0117] The nucleotide sequence encoding the second heavy chain variable region (T508-A1T-ACTA2-VH2) is shown below: CAGGTGAAGCTGCAGCAGTCTGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATGTGCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTTCGAATGATTATGCTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACATCAGACACATCCTCCAACACAGCCTACCTGCAGGTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGGTCTTTACTACGGTCGTAGCATTGACTACTGGGGCCAAGGCACCACTCTCACTGTCTCCTCAG (SEQ ID NO.2);

[0118] The nucleotide sequence encoding the third heavy chain variable region (T508-A1T-ACTA2-VH3) is shown below: GAGGTCAAGCTGCAGCAGTCTGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATGTGCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTTCGAATGATTATGCTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACATCAGACACATCCTCCAACACAGCCTACCTGCAGGTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGGTCTTTACTACGGTCGTAGCATTGACTACTGGGGCCAAGGCACCACTCTCACTGTCTCCTCAG (SEQ ID NO.3);

[0119] The nucleotide sequence encoding the fourth heavy chain variable region (T508-A1T-ACTA2-VH4) is shown below: CAGGTGCAGCTGCAGGAGTCTGGGACTGAGCTGGCAAGCCCTGGGGCTTCAGTGAAGTTGTCCTGTAAGGCTTCTGGCTACATCTTTTCTGACTATTGGATGCAGTGGGTAAAAGAGAGGCCTGGACAGGGTCTGGAATGGATTGGGACTATTTATCCTGGAGATGGTGATGCTCGATACAATCTGAAGTTCAAGAACAAGGCCGCTTTGACTGCAGATAAATCTTCCAATACAGCCTACATGTTGCTCAACAACTTGGCATCTGAAGACTCTGCGGTCTATTACTGTGCAAGATACTGGGATTACGACGTTGGGGGGTTTACTTACTGGGCCAGGGGTCTCTGGTCACTGTCTCTGCAG (SEQ ID NO.4).

[0120] According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (T508-A1T-ACTA2-VH1) is as follows:

[0121] EVKLQQSGAELVKPGASVRLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEINPNNGRTNYNEKFKSKATLTVDKSSRTAYMQLSSLTSEDSAVYYCAVIPTGYFDYWGQGTTLTVSS(SEQ ID NO.6);

[0122] The amino acid sequence of the second heavy chain variable region (T508-A1T-ACTA2-VH2) is as follows:

[0123] QVKLQQSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPSNDYAKYDPKFQGKATITSDTSSNTAYLQVSSLTSEDTAVYYCGLYYGRSIDYWGQGTTLTVSS(SEQ ID NO.7);

[0124] The amino acid sequence of the third heavy chain variable region (T508-A1T-ACTA2-VH3) is as follows:

[0125] EVKLQQSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPSNDYAKYDPKFQGKATITSDTSSNTAYLQVSSLTSEDTAVYYCGLYYGRSIDYWGQGTTLTVSS(SEQ ID NO.8);

[0126] The amino acid sequence of the fourth heavy chain variable region (T508-A1T-ACTA2-VH4) is as follows:

[0127] QVQLQESGTELASPGASVKLSCKASGYIFSDYWMQWVKERPGQGLEWIGTIYPGDGDARYNLKFKNKAALTADKSSNTAYMLLNNLASEDSAVYYCARYWDYDVGGFTYWGQGSLVTVSA (SEQ ID NO. 9).

[0128] Example 3: Expression screening of recombinant monoclonal ACTA2

[0129] 1. Small-scale eukaryotic expression of Expi CHO-S cells

[0130] Four recombinant monoclonal antibodies were paired with light and heavy chains in the manner of VK1 / VH1(R1), VK1 / VH2(R2), VK1 / VH3(R3), and VK1 / VH4(R4), respectively. They were then transfected and purified in 2 mL eukaryotic trials using the ExpiCHO-S™ Expression System.

[0131] The specific transfection method based on the ExpiCHO-S™ Expression System is as follows:

[0132] Select a density of 3×10 6 ~1×10 7 Seed cells with Expi CHO-S viable cells / mL and cell viability greater than 95% were used for transfection; the reagent required for transfection was calculated based on the volume of the cell suspension, so that the final cell density in each 2 mL transfection system was 3 × 10⁶ cells / mL. 6 Live cells / mL were incubated at 37°C with appropriate humidity (8% CO2) in a vibrating incubator at 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 × 10⁶ cells / mL. 61 live cells / mL, total volume 2 mL, seeded into a 6-well plate, with one positive control; dilute 4 µg plasmid DNA with 100 µL NaCl solution, and dilute 10 µg PEI solution with 100 µL NaCl solution. Add the diluted plasmid dropwise to each well using a pipette, add one positive control, return to the shaker, and incubate for 5 min. Then add the diluted PEI solution dropwise to each well, gently shake the 6-well plate, and return to the shaker for incubation at 37°C with appropriate humidity, 8% CO2, and a rotation speed of 125 rpm. 24 hours after transfection, add 50 µL sodium butyrate solution and 100 µL EN3 solution to each well. After 72 h, start sampling from the culture medium to detect recombinant protein expression.

[0133] Purification was performed using pure beans, and the specific steps are as follows:

[0134] Transfer 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. Centrifuge eukaryotic transfected cells at 1000 rpm for 5 min, collect 40 µL of supernatant, take an appropriate amount of precipitate, and dilute 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, and gradually transfer the supernatant to a new 1.5 mL EP tube, centrifuge, and collect 40 µL of supernatant as flow-through sample, discarding the remaining flow-through. Repeat this step to collect all beads. Add 10 µL of 5×Loading Buffer to the supernatant, precipitate, and flow-through, and add 20 µL of 5×Loading Buffer to the beads. Incubate all samples at 100℃ for 10 minutes. Boil for 1 minute and then perform SDS-PAGE gel electrophoresis for detection.

[0135] The results are as follows Figure 8 As shown, after eukaryotic transfection and antibody expression purification using the ExpiCHO-S™ Expression System (2 mL), the reduced SDS-PAGE electrophoresis showed clear bands at 25 kDa and 55 kDa without any extraneous bands, consistent with the expected target bands.

[0136] 2. Preparation of ACTA2 protein

[0137] The primer sequences for ACTA2 were designed using the 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 Biotechnology. 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 verification by agarose gel electrophoresis, and the target fragment of ACTA2 was recovered using the Tiangen General Agarose Gel DNA Recovery Kit (catalog number: DP209). The expression vector pET21b was digested with restriction endonucleases BamHI and XhoI. The target fragment of ACTA2 was then recombined with the linearized vector and transformed into competent BL21 cells (preserved in our laboratory). Single white colonies were picked for sequence identification. The identification results are as follows: Figure 7 Figure A (ACTA2 colony PCR agarose gel electrophoresis diagram) is shown.

[0138] The correctly identified clone was cultured in Luria-Bertani LB medium containing ampicillin, followed by protein expression induction at 37°C with 0.5 mmol / L IPTG (isopropyl-β-D-thiogalactopyranoside). The bacterial cells were collected, sonicated, and the supernatant was collected. The supernatant was added to nickel affinity packing material at a controlled flow rate, followed by elution with imidazole eluent at concentrations of 20 mmol / L, 50 mmol / L, 100 mmol / L, 250 mmol / L, and 500 mmol / L. 20 µL of the flow-through and different concentrations of imidazole eluent were subjected to denaturing and reducing polyacrylamide gel electrophoresis. The protein eluted with 50 mmol / L imidazole eluent was concentrated and subjected to non-denaturing, non-reducing polyacrylamide gel electrophoresis. The electrophoresis results are shown below. Figure 7 Figure B (ACTA2 polyacrylamide gel electrophoresis diagram) is shown.

[0139] 3. ELISA detection of the titer of mouse anti-human ACTA2 recombinant monoclonal antibody

[0140] To further screen for the optimal mouse anti-human ACTA2 recombinant monoclonal antibody sequence, a 2 mL eukaryotic transfection and antibody expression purification were performed using the ExpiCHO-S™ Expression System, followed by ELISA detection of the supernatant titer.

[0141] The specific methods for determining potency are as follows:

[0142] 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 microplate, and then react overnight at 4ºC.

[0143] 2) Washing the plate: Pour out the liquid from the 96-well microplate, wash the plate 3 times with PBST buffer, and then pat the liquid in the 96-well microplate dry on filter paper;

[0144] 3) Blocking: Add 200 µL of 2% BSA (bovine serum albumin) to each well and incubate at 37°C for 2 hours;

[0145] 4) Washing the plate: Pour out the liquid from the 96-well microplate, wash the plate 3 times with PBST buffer, and then pat the liquid in the 96-well microplate dry on filter paper;

[0146] 5) Add primary antibody: Set antibody concentrations of 10 ng / mL, 3.33 ng / mL, and 1.11 ng / mL. Dilute the antibody according to these three concentration gradients. Add 100 µL of diluted antibody (mouse anti-human ACTA2 recombinant monoclonal antibody prepared in this invention) to each well and incubate at 37°C for 1 hour.

[0147] 6) Washing the plate: Pour out the liquid from the 96-well microplate, wash the plate 3 times with PBST buffer, and then pat the liquid in the 96-well microplate dry on filter paper.

[0148] 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;

[0149] 8) Washing the plate: Pour out the liquid from the 96-well microplate, wash the plate 3 times with PBST buffer, and then pat the liquid in the 96-well microplate dry on filter paper;

[0150] 9) Color development: Add 100 µL of color development solution to each well, place in an incubator, incubate at 37℃ for 10 min, and add 50 µL of 2mol / L sulfuric acid solution to terminate the reaction;

[0151] 10) OD value determination: Use an ELISA reader to determine the absorbance (OD 450) value of each well.

[0152] The results showed that the expression of recombinant antibodies paired with T508-A1T-ACTA2-VK1 / VH2 and T508-A1T-ACTA2-VK1 / VH3 was as follows: Figure 8As shown, A represents the expression of T508-A1T-ACTA2-VH2 / VK1 and T508-A1T-ACTA2-VH1 / VK1, with band 4 representing T508-A1T-ACTA2-VH2 / VK1 and band 8 representing T508-A1T-ACTA2-VH1 / VK1; B represents the expression of T508-A1T-ACTA2-VH3 / VK1 and T508-A1T-ACTA2-VH4 / VK1, with band 8 representing T508-A1T-ACTA2-VH3 / VK1 and band 12 representing T508-A1T-ACTA2-VH12 / VK1. The ELISA results for T508-A1T-ACTA2-VK1 / VH3 showed a weak positive result, while T508-A1T-VK1 / VH2... The ELISA result was positive, with a strong signal, indicating good recognition of coated ACTA2 proteins. Figure 11 As shown in the figure. Figure A shows the titer results of T508-A1T-ACTA2-VH1 / VK1(R1), B shows the titer results of T508-A1T-ACTA2-VH2 / VK1(R2), C shows the titer results of T508-A1T-ACTA2-VH3 / VK1(R3), and D shows the titer results of T508-A1T-ACTA2-VH4 / VK1(R4).

[0153] The T508-A1T-VK1 / VH2 bacterial culture was sent to a sequencing company for sequencing analysis. The sequencing results are as follows: Figure 9 As shown, the results indicate that the sequencing peaks are distinct, clear, and symmetrical, with good resolution and signal-to-noise ratio, and no abnormal or extraneous peaks, indicating that the sequencing results are reliable and can be used for subsequent experiments.

[0154] To obtain milligram-level recombinant ACTA2 antibodies for sample validation and characterization, this study utilized the ExpiCHO-S™ Expression System to perform 60 mL eukaryotic pilot-scale expression and antibody affinity chromatography purification of the screened T508-A1T-ACTA2-VK1 / VH2 antibody. The purified recombinant antibody was then analyzed by reducing SDS-PAGE (e.g., ...). Figure 10 As shown in the figure, there are clear and distinct bands at 25 kDa and 55 kDa without any extraneous bands, which is consistent with the expected target band.

[0155] Example 4:

[0156] This embodiment describes the ELISA affinity detection of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the methods described in Examples 1-3. The detection process is as follows:

[0157] To further characterize the affinity of the recombinant ACTA2 antibody, an indirect ELISA method was used for identification, followed by OD...450 Using nm as the ordinate and the logarithm of antibody concentration as the abscissa, two curves were fitted using Origin 8.5 software to calculate the OD. max The antibody affinity constant Ka was calculated based on the antibody concentration (mol / L) corresponding to / 2. The results are as follows: Figure 12 As shown, A represents the concentration of the coated recombinant protein as 0.5 μg / mL, and B represents the concentration of the coated recombinant protein as 1 μg / mL.

[0158] Calculated based on the affinity constant Ka (Equation 1-1):

[0159] Ka = (n-1) / 2( n[Ab]1-[Ab]2) (Formula 1-1)

[0160] In the formula, [Ab]1 and [Ab]2 represent two different coating concentrations at OD. max The antibody concentration (mol / L) corresponding to / 2 is [Ab]1, which corresponds to a relatively low coating concentration, and [Ab]2, which corresponds to a relatively high coating concentration. n is the ratio of the two coating concentrations (n>1).

[0161] The affinity of the variable region coding gene of the recombinant monoclonal antibody against ATCA2 was calculated, and the results showed that the affinity of the variable region coding gene of the recombinant monoclonal antibody against ATCA2 was 6.6 × 10⁻⁶. 10 L / mol, affinity greater than 10 7 L / mol indicates a high-affinity antibody, suggesting that the variable region encoding gene of the recombinant monoclonal antibody against ATCA2 prepared in this invention has high affinity.

[0162] Example 5:

[0163] This embodiment describes the Western blot detection of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the methods described in Examples 1-3. The detection process is as follows:

[0164] To verify the binding ability and specificity of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared in this invention to the natural ACTA2 antigen in cell samples, this embodiment performed Western blotting on the mouse anti-human ACTA2 recombinant monoclonal antibody and a commercial antibody (Leica). Bands were detected at corresponding molecular weight positions for Human colon, Human spleen, Rar brain, Rar heart, Mouse heart, Mouse brain, 293T, A431, MCF-7, HepG2, and K-562 (preserved in our laboratory). The results showed that the ACTA2 protein in positive cells had specific recognition ability, while no target band was found in Human colon, indicating a negative result.

[0165] The results are as follows Figure 13 As shown in the figure, the results showed no statistically significant difference in ACTA2 protein levels among the cells tested (p>0.05). This indicates that the recombinant ACTA2 antibody prepared in this study can bind to the common epitopes of mutant and non-mutant ACTA2 proteins, exhibiting good specificity and sensitivity in Western blotting experiments.

[0166] Example 6:

[0167] This embodiment describes the immunohistochemical detection of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared according to the methods described in Examples 1-3. The detection process is as follows:

[0168] To further verify the recognition ability of the mouse anti-human ACTA2 recombinant monoclonal antibody prepared in this invention on ACTA2 protein expression in pathological tissue samples, this embodiment performed immunohistochemical verification on more than 40 tissue samples. The specific steps are as follows:

[0169] (1) Prepare tissue paraffin blocks. Seven types of human cancer tissue paraffin blocks were selected, namely rectal adenocarcinoma, liver cancer, breast cancer, well-differentiated squamous cell carcinoma, phyllodes carcinoma of the breast, renal cancer, sigmoid colon adenocarcinoma, and three types of animal tissue paraffin blocks, namely mouse liver cancer and normal tissue (testis and duodenum) of monkey. Four consecutive sections were taken from each type of tissue.

[0170] (2) The detection was performed using commercially available ACTA2 antibody and self-developed ATCTA2 recombinant antibody, respectively;

[0171] (3) Place the paraffin sections in a 60℃ oven for 60 min, and then dewax and hydrate them using a Leica multi-functional staining machine;

[0172] (4) EDTA high-temperature antigen retrieval: Take a certain amount of pH 9.0 EDTA antigen retrieval working solution into the retrieval box. The amount of retrieval solution must be sufficient to submerge the entire slide. Place the retrieval box into a pressure cooker with an appropriate amount of tap water added. Heat over high heat until boiling. Place the dewaxed and hydrated tissue slide on a high-temperature staining rack. Then slowly place the staining rack into the retrieval box. Cover the pot and do not close the pressure valve. Start timing for 20 minutes. After that, disconnect the power to the pressure cooker, remove the valve and open the lid. Take out the retrieval box and let it cool naturally at room temperature for 20 minutes before taking out the slide.

[0173] (5) Rinse twice with pure water for 5 min each time, add 3% H2O2, and incubate in a humidified box at room temperature for 20 min;

[0174] (6) Rinse three times with PBS buffer, 5 min each time. Add sheep serum for blocking and incubate in a humidified chamber at 37°C for 30 min;

[0175] (7) Discard the blocking solution, add the primary antibody diluted at a ratio of 1:100 and 1:200, incubate at 4°C overnight, and then warm at 37°C for 30 min;

[0176] (8) Discard the primary antibody and soak in PBS buffer 3 times, 5 min each time;

[0177] (9) Add goat anti-mouse secondary antibody, incubate in a humidified chamber at 37°C for 30 min, and discard the secondary antibody;

[0178] (10) Soak in PBS buffer 3 times, 5 min each time, add DAB working solution, observe the color development under a microscope to control the time, and rinse with pure water after the color development is complete.

[0179] (11) Counterstain with hematoxylin for 3 s, rinse with tap water for 5 min, soak in 1% hydrochloric acid ethanol for 1 s, and soak in tap water for 2 min;

[0180] (12) Stain with Scott's for 2 min, soak in tap water for 2 min, and examine under a microscope;

[0181] (13) Use a Leica multi-functional staining machine to dehydrate, clear, and seal the slides, and finally take pictures and scan the slides.

[0182] Some results are as follows Figure 14 As shown. Among them Figure 14 A shows the results of ACTA2 recombinant antibody immunohistochemical detection in human breast cancer / human rectum; B shows the results of ACTA2 recombinant antibody immunohistochemical detection of subcellular localization in human liver IF; C shows the results of ACTA2 recombinant antibody immunohistochemical detection of subcellular localization in human smooth muscle IF; D shows the results of ACTA2 recombinant antibody immunohistochemical detection of cell localization and subcellular localization using A549 confocal imaging and 64x imaging.

[0183] The results showed that ACTA2 was selectively expressed in smooth muscle cells and myoepithelial cells, mainly localized to actin filaments. This antibody specifically bound to the target antigen, consistent with the expected tissue, tissue sub-localization, cellular, and subcellular localization; and the signal was strong, suggesting good application in IHC. ACTA2 is mainly localized to actin filaments. The results showed that this antibody specifically bound to the target antigen, consistent with the expected tissue, tissue sub-localization, cellular, and subcellular localization; and the signal was strong, suggesting good application in IF. ACTA2 is mainly localized to actin filaments. The results showed that this antibody specifically bound to the target antigen, consistent with the expected cellular and subcellular localization; and the signal was strong, suggesting good application in ICC.

[0184] In summary, this invention successfully prepared a mouse anti-human ACTA2 recombinant monoclonal antibody with high titer and affinity. The Western blot and immunohistochemical detection results of the recombinant ACTA2 antibody were similar to those of commercially available ACTA2 antibodies. In some tissue samples, the positive rate of immunohistochemical detection was significantly higher than that of commercially available ACTA2 antibodies. This antibody can be used for Western blot and immunohistochemical detection and can provide preliminary data support for clinical pathological diagnosis.

[0185] Finally, it should be emphasized that the above description 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 can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant monoclonal antibody against ATCA2, characterized in that, The recombinant monoclonal antibody against ATCA2 includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 6; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

10.

2. The encoding gene of the recombinant monoclonal antibody against ATCA2 according to claim 1, characterized in that, The encoding gene includes the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.

5.

3. The encoding gene according to claim 2, 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 coding 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 coding gene of the recombinant monoclonal antibody against ATCA2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene as described in any one of claims 2-3.

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

6. A recombinant expression cell, characterized in that, The recombinant expression cell contains the coding gene as described in any one of claims 2-3, or contains the recombinant expression vector as described in any one of claims 4-5.

7. A method for preparing the recombinant monoclonal antibody against ATCA2 as described in claim 1, characterized in that, The preparation method is as follows: recombinant expression cells are obtained by transfecting cells with the recombinant expression vector as described in claim 4 or 5, and the recombinant expression cells are obtained after culture and transfection. The supernatant is collected and purified to obtain a recombinant monoclonal antibody against ATCA2.

8. The use of the encoding gene as described in any one of claims 2-3, or the recombinant expression vector as described in any one of claims 4-5, or the recombinant expression cell as described in claim 6 in the preparation of the recombinant monoclonal antibody against ATCA2 as described in claim 1.

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