Anti-smooth muscle myosin monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a monoclonal antibody capable of recognizing a human smooth muscle ball antigen, a preparation method and application of the monoclonal antibody in immunodetection. According to the technical scheme, 1180-1400 amino acids of smooth muscle myosin are selected as antigen peptides, codon optimization is carried out, a gene segment suitable for being expressed in escherichia coli is formed, and finally the obtained recombinant protein contains a smooth muscle myosin segment and a histidine protein tag. A mouse is immunized by the recombinant protein, and is subjected to cell fusion, screening and subcloning to obtain a mouse hybridoma cell strain 15C1D4 and an anti-smooth muscle myosin monoclonal antibody secreted by the cell strain. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing smooth muscle myosin, and is suitable for immunological detection, especially immunohistochemical detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to an anti-smooth muscle myosin monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Myosin, Smooth Muscle, is a protein that plays a key role in smooth muscle cells, especially its heavy chain, which is crucial for the contractile function of smooth muscle. The smooth muscle myosin heavy chain is a polypeptide with a molecular weight of 200kDa and is a structural component of hexameric myosin. It is mainly expressed in smooth muscle tissues, which are widely distributed in multiple systems of the human body, including but not limited to the digestive system, respiratory system, urinary system, and reproductive system, and maintain the normal physiological functions of the human body through contraction and relaxation functions.

[0003] Myosin, Smooth Muscle is expressed in many tumors, but the specific expression varies depending on the tumor type and stage. Smooth muscle myosin heavy chain is often used in the diagnosis and classification of mesenchymal tumors. It can be used as an important marker to distinguish leiomyosarcoma from other types of mesenchymal tumors. In breast tumors, smooth muscle myosin heavy chain can be used to detect breast myoepithelial cells, which helps to distinguish breast carcinoma in situ from invasive carcinoma. At the same time, the detection of smooth muscle myosin heavy chain is also one of the auxiliary means of tumor diagnosis. The type and origin of the tumor can be preliminarily determined by detecting the expression level of smooth muscle myosin heavy chain in tumor tissue or cell samples. Myosin, Smooth Muscle, as an important protein component in smooth muscle cells, has broad application prospects in the diagnosis, treatment and prognosis evaluation of tumors. Summary of the invention

[0004] The inventors provide an anti-smooth muscle myosin monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.

[0005] Furthermore, the monoclonal antibody is a mouse monoclonal antibody with a clone number of 15C1D4.

[0006] The inventors have also provided a method for preparing an anti-smooth muscle myosin monoclonal antibody, wherein the antigen used to immunize mice is a recombinant protein, and the recombinant protein is recombinantly expressed by Escherichia coli.

[0007] Furthermore, the recombinant protein comprises a smooth muscle myosin fragment and a HIS protein tag.

[0008] Furthermore, the smooth muscle myosin fragment is a fragment of positions 1180-1400, which is the amino acid sequence shown in SEQ ID NO.3.

[0009] Furthermore, the plasmid vector used in the recombination process was pET30a.

[0010] The inventors also provide a smooth muscle myosin immunoassay reagent, which contains the above anti-smooth muscle myosin monoclonal antibody as its effective ingredient.

[0011] Furthermore, the immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

[0012] Different from the prior art, the beneficial technical effect produced by the present invention is as follows: the above technical scheme selects the amino acids at positions 1180-1400 of smooth muscle myosin as antigenic peptides, performs codon optimization, and becomes a gene fragment suitable for expression in Escherichia coli BL21 (DE3), and the recombinant protein finally obtained contains a smooth muscle myosin fragment and a histidine protein tag. The recombinant protein is used to immunize mice, and after cell fusion, screening and subcloning, a mouse hybridoma cell line 15C1D4 and an anti-smooth muscle myosin monoclonal antibody secreted by the cell line are obtained. The antibody obtained by this scheme has high specificity and sensitivity, can specifically identify cells expressing smooth muscle myosin, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure is a comparison chart of breast cancer immunohistochemical staining results; the left one is the antibody prepared by the present invention, and the right one is a commercially available antibody.

[0014] Figure 2 The figure is a comparison of the results of immunohistochemical staining of the intestinal surface epithelium; the left one is the antibody prepared by the present invention, and the right one is a commercially available antibody. DETAILED DESCRIPTION

[0015] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0016] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0017] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0018] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0019] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0020] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0021] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0022] Example 1 Preparation of recombinant smooth muscle myosin fragments

[0023] 1. Gene Optimization and Synthesis

[0024] According to the protein sequence of the smooth muscle myosphere with accession number XP_063479755.1 in the NCBI database, the protein fragment of position 1180-1400 was selected and directly optimized into a gene fragment suitable for expression in Escherichia coli BL211 (DE3). During the PCR process, BamH I and XhoI restriction sites were added to the 5' and 3' ends of the gene respectively.

[0025] The PCR products were recovered after separation by agarose gel electrophoresis, and the recovered fusion protein gene and the plasmid vector pET30a used for expression were digested by BamH I and XhoI, respectively, recovered by electrophoresis again, and connected with T4 DNA ligase. The ligation products were transformed into Escherichia coli competent cells BL211 (DE3), and the clones on the plate were picked and inoculated for bacterial liquid PCR identification. The clones with positive PCR results were selected for sequencing analysis, and the clones with completely correct sequences were used.

[0026] Choosing different antigens for immunization may produce antibodies with different binding properties. The molecule also has multiple variants caused by variable splicing, which ultimately leads to different recognition abilities and patterns of different antibodies for antigen-expressing cells. The smooth muscle myosin molecule was analyzed according to the published sequence. Based on its structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure, the region suitable for soluble expression and good immunogenicity was selected for recombinant expression. The amino acid residues 1180-1400 of the smooth muscle myosin were selected for codon optimization, and the molecular weight was about 220kDa. Smooth muscle myosin was obtained by sequence optimization design using the prokaryotic expression gene sequence. The recombinant immunogen consists of an antigenic smooth muscle myosin fragment and a protein tag for recombinant protein purification, and the protein tag is HIS.

[0027] 2. Protein Expression and Purification

[0028] Transfer the overnight bacteria cultured from a single colony to 100mL LB medium at a ratio of 1:100, add kanamycin with a final concentration of 10μg / mL, shake culture at 37℃ until OD600 is 0.6-0.8, add 0.5mmol / L IPTG, shake culture at 16℃ overnight, and ultrasonically break the bacteria after harvesting. The recombinant protein has a histidine tag, and a nickel column is used for affinity purification of the protein. Elution is performed with 500mmol / L imidazole, and SDS PAGE separation and detection are performed. The concentration of recombinant smooth muscle myosin fused with histidine tag is 0.5mg / mL, which can be used for animal immunization and antibody screening and identification requirements.

[0029] Example 2 Establishment of hybridoma cell lines

[0030] 1. Immunity

[0031] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881) and immunized 4-6 week old female ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and injected subcutaneously at 6 points in the abdomen of each mouse at a dose of 20 μg / mouse. The booster immunization was performed once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg / mouse. Seven days after the third booster immunization, the polyantibody titer against the immunogen in the mouse serum was detected by indirect ELISA (wavelength 450nm). The mouse with the highest titer was immunized by tail vein injection, and the antigen was mixed with physiological saline at a dose of 20 μg / mouse.

[0032] 2. Cell Fusion

[0033] Prepare aseptically the spleen cell suspension of immune-qualified mice, mix it with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After discarding the supernatant, place the centrifuge tube in a 37°C water bath, slowly add 1mL of PEG1500 (Roche) within 1 minute, and stir the cells. After standing in warm water for 1min, add 10mL of serum-free IMDM (Sigma), mix well, and centrifuge at 1000rpm for 5min. After discarding the supernatant, add 10mL of serum (PAA) and carefully blow up the cells, and add 5mL of thymocytes mixed with 10xHAT (Sigma) and mix well. Then add 25mL of semi-solid culture medium containing 2.1% nitrocellulose (Sigma) and mix well, then pour evenly into 20 cell culture dishes. Place the cell culture dishes in a humidified box and place them in 37°C 5% CO 2 Culture in an incubator.

[0034] 3. Cloning and ELISA screening of positive hybridoma cells

[0035] Seven days after fusion, the size and density of cloned cell clusters were moderate. Under the dissecting microscope, round, solid, and large clones were picked up and placed into a 96-well culture plate prepared with culture medium in advance and placed in a 37°C 5% CO 2 Culture in an incubator. After 3 days, the amount of cells accounts for about 2 / 3 of the bottom area. Take 100 μL of supernatant and perform ELISA screening with immunogen and synthetic peptide respectively. Replace the medium for positive clones completely, and add 200 μL of complete culture medium containing feeder cells and 1% HT (Sigma). Two days later, perform a second ELISA screening, and transfer positive clones to 24-well plates with culture medium (containing feeder cells and HT) prepared in advance for culture. Five days later, take 100 μL of supernatant for the third ELISA screening, and transfer positive clones to 6-well plates and cell culture flasks for expansion and cryopreservation.

[0036] Example 3 Preparation of monoclonal antibodies by ascites induction method

[0037] 1. Ascites Preparation

[0038] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, and approximately 5 × 10 5 , 1mL. The suspended cells were intraperitoneally injected into mice sensitized with paraffin oil in advance. Ascites was collected 7 days later. The ascites was centrifuged at 4000rpm for 10min at 4℃. The ascites in the middle was carefully aspirated and collected in a centrifuge tube and stored at 4℃ or -20℃.

[0039] 2. Purification of Monoclonal Antibodies

[0040] The antibody was purified from ascites using HiTrap rProtein A FF (GE) affinity chromatography according to the instructions. The purity was determined by SDS-PAGE gel and the concentration was determined by Bradford method. The purified antibody was stored at -20°C.

[0041] Example 4 Monoclonal Antibody Characterization

[0042] 1. Subclass Identification

[0043] Dilute the coated goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to 0.5μg / mL with 100mM PBS (pH7.4), add 100μL to each well, and incubate at 4℃ overnight. Pour out the liquid, wash 3 times with PBS containing 0.05% Tween (PBS-T), add 200μL blocking solution (PBS containing 2% BSA and 3% sucrose) to each well, and incubate at 37℃ for 1h. Pour out the liquid, wash 3 times with PBS-T. Add 0.1mL hybridoma supernatant to each well, and incubate at 37℃ for 1h. Pour out the liquid and wash 3 times with PBS-T. Use blocking solution to dilute HRP-labeled goat anti-mouse (κ, λ) antibody at 1:1000 or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) at 1:2000, add 0.1 mL to each well and incubate at 37°C for 1 hour. Pour out the liquid and wash three times with PBS-T. Add 50 μL of 0.15% ABTS (Southern Biotech) and 0.03% H 2 O 2 The color reaction was carried out with citric acid buffer (PH 4.0), and the OD value at a wavelength of 405 nm was measured within 10-20 min.

[0044] The results showed that the monoclonal antibody of the present invention is an IgG2b type mouse monoclonal antibody.

[0045] 2. Determination of affinity constant

[0046] The smooth muscle myosin recombinant protein prepared in Example 3 was coated at a concentration of 2 μg / mL, 100 μL / well, coated overnight at 4°C, and washed 3 times with PBS-T. 200 μL of blocking solution was added to each well and blocked at 37°C for 2 hours, and washed 3 times with PBS-T. The monoclonal antibody purified in Example 3 was diluted 2-fold starting from 1:200, and a blank control was left in the last well, incubated at 37°C for 1 hour, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody was diluted 1:20000, 100 μL per well, incubated at 37°C for 1 hour, and washed 3 times with PBS-T. 100 μL of 0.1% TMB (Sigma) and 0.03% H was added to each well. 2 O 2 The color was developed with citric acid-phosphate buffer for 10 minutes, and 50 μL of 0.5 M sulfuric acid solution was added to terminate the reaction. The absorbance at a wavelength of 450 nm was measured using an enzyme marker. A curve of OD value versus antibody dilution multiple was drawn, and the dilution multiple A corresponding to half of the maximum binding OD value was found. The affinity constant of the antibody was calculated using the following formula: 1.92×10 9 .

[0047]

[0048] 3. Monoclonal antibody reaction specificity and application effect

[0049] Take the smooth muscle myosin recombinant protein prepared in Example 1, use the immunoblotting method to detect the recognition specificity of the monoclonal antibody of the present invention, and perform 12% polyacrylamide gel electrophoresis. Transfer the gel protein band to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to conventional methods. Place the membrane in a TBS-T blocking solution containing 5% skim milk powder at 4°C overnight. Add the smooth muscle myosin monoclonal antibody purified in Example 3 (1:1000 dilution) and incubate at 4°C overnight. After washing the membrane with TBS-T, add a 1:5000 diluted goat anti-mouse secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) and incubate at room temperature for 1 hour. Wash the membrane again with TBST, add ECL ultrasensitive colorimetric solution (Beijing Pulilai Gene Technology Co., Ltd.), and use the ChemiDocMP multicolor fluorescence imaging system (Bio-Rad) to collect chemiluminescent image data.

[0050] Example 5 Determination of variable region sequences of antibodies

[0051] Take freshly cultured hybridoma cells and take the supernatant to verify the antigen binding characteristics to confirm that the cell line used for cloning can indeed secrete the required antibodies. After the results are confirmed, centrifuge and collect for 10 6The above hybridoma cells. Trizol method was used to extract total RNA of hybridoma cells. 9 μL of total RNA was taken, 2.5 μL of oligo(dT)12–18primer (10 mM) and 5 μL of dNTPs were added, mixed evenly, kept at 70°C for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Subsequently, 5 μL of RTbuffer (5X), 2.5 μL of DTT (0.1 M) and 1 μL of reverse transcriptase were added, and the reaction was carried out at 42°C for 1 hour. The reaction was terminated by incubation at 70°C for 15 minutes, and the obtained cDNA was stored at -20°C. The first-strand cDNA obtained was subjected to PCR amplification, and 25 pmol of each primer was added to the 50 μL reaction system. The sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized according to the mouse monoclonal antibody primer sequences in the book "Recombinant Antibodies" edited by Shen Beifen (Science Press, published in 2005).

[0052] The remaining dNTPs and buffer were added as usual, and finally 1 μL of cDNA template and 1U hot start Taq DNA polymerase were added. The PCR amplification program was set to 94°C for 40 seconds, 52°C for 40 seconds, and 72°C for 40 seconds for 20 to 25 cycles, and finally extended at 72°C for 3 minutes. The product can be placed at 4°C for standby or directly electrophoresed. Take 20 μL of PCR product for electrophoresis analysis and separate on 1.5% agarose gel. The length of the light chain (κ light chain) is between 320-340 bp, and the length of the heavy chain is between 340-370 bp. When there is a specific product in this region, the gel is cut and recovered, cloned into a T vector or expression vector for sequencing.

[0053] Example 6. Immunohistochemical tissue chip staining and identification

[0054] 1. Chip preparation process

[0055] Each sample was first stained with HE slices to determine the tumor site. The tissue chip was made using the fully automatic tissue chip instrument of 3DHISTECH. The prepared tissue chip wax block was placed in the wax block mold, placed in a 68°C oven for 10 minutes, so that the tissue core and the wax of the receptor wax block were integrated, and then the mold was gently removed from the oven, and the semi-melted paraffin was allowed to cool at room temperature for about 30 minutes, and then placed in a -20°C refrigerator for 6 minutes. After the tissue chip wax block was removed from the mold, sliced ​​or placed in a 4°C refrigerator for later use. After trimming, continuous sections were made with a thickness of 3μm. The continuous sections were floated in 40% alcohol and allowed to unfold naturally. The separated sections were then transferred to 50°C warm water for 30 seconds, and the sections were mounted on slides treated with polylysine. The prepared tissue chip was placed in a 68°C oven for 2 hours, taken out, cooled at room temperature, and stored in a -4°C refrigerator.

[0056] II. IHC staining and analysis

[0057] Dewax in xylene for 3 times, 6 minutes each time, hydrate in 100%, 100%, 95%, 85% graded ethanol, 3 minutes each time, and finally rinse with tap water. Perform antigen repair, then place the sections in a wet box and rinse with PBS for 3×3 minutes. Add 3% H 2 O 2 Incubate for 10 minutes, rinse with PBS for 3×3 minutes. Shake dry the slices, add the primary antibody diluted in an appropriate ratio (the dilution ratio of the antibody is designed according to the antibody concentration for the first dilution), incubate at room temperature (25°C) for 1 hour, rinse with PBS for 3×3 minutes, add the secondary antibody and incubate at room temperature for 15-30 minutes, rinse with PBS for 3×3 minutes, shake off the PBS, and use freshly prepared DAB color developing solution for 3-10 minutes. Counterstain with hematoxylin for 25 seconds, and return to blue with PBS for 30 seconds. Dehydrate in a gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes) alcohol, and finally transparentize with xylene for 3 minutes and seal with neutral gum.

[0058] Immunohistochemical staining results are divided into: positive and negative. Positive expression must be in specific antigenic sites of cells and tissues to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0059] 1. The sample is weakly positive; marked as "+";

[0060] 2. The sample is moderately positive; marked as "++";

[0061] 3. The sample is highly positive; marked as "+++".

[0062] 4. The sample is negative and marked as “-”.

[0063] 3. Data Statistics

[0064] 1. Tumor tissue chip test results:

[0065] The antibody smooth muscle myosin (15C1D4) and the commercially available antibody smooth muscle myosin antibody (SMMS-1) were used to simultaneously detect 62 cases of breast cancer and the detection results were compared.

[0066] The immunohistochemical results of the smooth muscle myosin monoclonal antibody were statistically analyzed. The entire experimental process adopted a double-blind design, and the statistical results are as follows:

[0067]

[0068] The results showed that the anti-smooth muscle myosin monoclonal antibody prepared by the present invention had accurate staining positioning, clear staining without nonspecific staining, and clean background. In immunohistochemical detection, the positive rate was comparable to that of commercially available antibodies, but there were 2 cases with higher positive intensity than commercially available antibodies. This shows that the smooth muscle myosin prepared by the present invention has higher sensitivity and effectively avoids false negative results.

[0069] Figure 1 The figure is a comparison chart of breast cancer immunohistochemical staining results (the left one is the antibody prepared by the present invention, and the right one is a commercially available antibody).

[0070] 2. Normal tissue chip test results:

[0071] The normal tissue array includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid gland, liver, pituitary gland, salivary gland, testis, kidney, thyroid gland, prostate gland, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infants), skin, bone marrow, peripheral nerve, lung, and mesothelial cells.

[0072] The present antibody (15C1D4) and commercially available antibodies were simultaneously tested on normal tissue chips, and the positive and negative test results were consistent, indicating that the specificity of the present antibody in normal tissues is equivalent to that of the commercially available antibodies. Figure 2 The figure is a comparison of the results of immunohistochemical staining of the intestinal surface epithelium (the left one is the antibody prepared by the present invention, and the right one is a commercially available antibody).

[0073] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. An anti-smooth muscle myosin monoclonal antibody, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody is a mouse monoclonal antibody with a clone number of 15C1D4.

3. A method for preparing an anti-smooth muscle myosin monoclonal antibody, characterized in that: The antigen used for immunizing mice is a recombinant protein, which is recombinantly expressed by Escherichia coli.

4. The preparation method according to claim 3, characterized in that: The recombinant protein comprises a smooth muscle myosin fragment and a HIS protein tag.

5. The preparation method according to claim 4, characterized in that: The smooth muscle myosin fragment is a fragment of positions 1180-1400, which has an amino acid sequence shown in SEQ ID NO.

3.

6. The preparation method according to claim 3, characterized in that: The plasmid vector used in the recombination process was pET30a.

7. A smooth muscle myosin immunoassay reagent, characterized in that: The immunoassay reagent contains the anti-smooth muscle myosin monoclonal antibody according to claim 1 as its effective ingredient.

8. The immunoassay reagent according to claim 7, characterized in that: The immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

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