Anti-smooth muscle myosin monoclonal antibodies, methods of making and uses thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human smooth muscle myosin antigen, preparation method and its use in immunodetection.The above technical solution selects the amino acid of 1180-1400 of smooth muscle myosin as antigen peptide, codon optimization becomes the gene fragment suitable for expression in escherichia coli, and finally the recombinant protein includes smooth muscle myosin fragment and histidine protein label.The recombinant protein is immunized to mouse, and is subjected to cell fusion, screening and subcloning, obtains mouse hybridoma cell strain 15C1D4 and the anti-smooth muscle myosin monoclonal antibody secreted by the cell strain.The antibody obtained by the present application has high specificity, sensitivity, and can specifically recognize the cell expressing smooth muscle myosin, and is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

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

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

[0003] Myosin, Smooth Muscle is expressed in a variety of tumors, but the specific expression varies depending on the type and stage of the tumor. Smooth muscle myosin heavy chain is often used for the diagnosis and classification of mesenchymal tumors. It can be used as an important marker for differentiating leiomyosarcoma from other types of mesenchymal tumors. In breast tumors, smooth muscle myosin heavy chain can be used for the detection of breast myoepithelial cells, which helps to differentiate 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 for tumor diagnosis. By detecting the expression level of smooth muscle myosin heavy chain in tumor tissue or cell samples, the type and origin of the tumor can be preliminarily judged. As an important protein component in smooth muscle cells, Myosin, Smooth Muscle has a wide application prospect in the diagnosis, treatment and prognosis evaluation of tumors. SUMMARY

[0004] The present application provides 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; and 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] Further, the monoclonal antibody is a murine monoclonal antibody, and the clone number is 15C1D4.

[0006] The present application further provides a preparation method of an anti-smooth muscle myosin monoclonal antibody, wherein the antigen for immunizing a mouse is a recombinant protein, and the recombinant protein is expressed by E. coli.

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

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

[0009] Further, the plasmid vector selected in the recombinant process is pET30a.

[0010] The inventor also provides a smooth muscle myosin immunodetection reagent, which contains the above-mentioned anti-smooth muscle myosin monoclonal antibody as an effective component.

[0011] Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunization.

[0012] Differing from the prior art, the beneficial technical effects of the present application are: the above technical solution selects the amino acid at site 1180-1400 of smooth muscle myosin as an antigenic peptide, which is codon-optimized to become a gene fragment suitable for expression in Escherichia coli BL21(DE3), and finally a recombinant protein containing a smooth muscle myosin fragment and a histidine protein tag is obtained. The recombinant protein is used to immunize mice, and after cell fusion, screening and subcloning, a mouse hybridoma cell strain 15C1D4 and an anti-smooth muscle myosin monoclonal antibody secreted by the cell strain are obtained. The antibody obtained by the present solution has high specificity and sensitivity, and can specifically recognize cells expressing smooth muscle myosin, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0016] The term "embodiment" is mentioned in this document means that the specific features, structures or characteristics described in connection with the embodiment can 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 the independence or association between other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

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

[0018] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this document generally represents that the associated objects before and after are a "or" logical relationship.

[0019] In this application, such as "first" and "second" language is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0020] In this application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent in such process, method or product.

[0021] As the same understanding as in the "Guidelines for Examination", in this application, "greater than", "less than", "exceed" and other expressions are understood as not including the number; "above", "below", "within" and other expressions are understood as including the number. In addition, the meaning of "multiple" in the description of the embodiments of the present application is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

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

[0023] I. Gene optimization and synthesis

[0024] The protein fragment of 1180-1400 of the smooth muscle myosin according to the protein sequence with accession number XP_063479755.1 in the NCBI database was directly optimized into a gene fragment suitable for expression in E. coli BL21 (DE3). BamH I and Xho I enzyme cutting sites were added at the 5' and 3' ends of the gene during PCR.

[0025] The PCR product was recovered after agarose gel electrophoresis separation, and the recovered fusion protein gene and the plasmid vector pET30a for expression were subjected to BamH I and Xho I enzyme cutting, respectively, and then recovered again after electrophoresis and linked with T4 DNA ligase. The ligation product was transformed into competent E. coli cells BL21 (DE3), and the clones on the plate were inoculated and subjected to 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] Different antigens selected for immunization can produce antibodies with different binding properties. The molecule contains multiple variants caused by variable splicing, which ultimately leads to different recognition abilities and patterns of different antibodies to cells expressing the antigen. According to the published sequence, the smooth muscle myosin molecule was analyzed, and based on its structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression. The amino acid residues of 1180-1400 of the smooth muscle myosin were selected for codon optimization, and the molecular weight was about 220 kDa. The smooth muscle myosin protein was obtained by designing the sequence of the prokaryotic expression gene through sequence optimization. The recombinant immunogen is composed of the smooth muscle myosin protein fragment with antigenicity and the protein tag for purification of the recombinant protein, and the protein tag is HIS.

[0027] II. Protein expression and purification

[0028] The single colony culture overnight bacteria were transferred to 100 mL of LB medium at a ratio of 1:100, kanamycin was added at a final concentration of 10 μg / mL, and the culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then, 0.5 mmol / L IPTG was added, and the culture was incubated at 16°C with shaking overnight. After harvesting the bacteria, they were sonicated. The recombinant protein has a histidine tag, and nickel column was used for affinity purification of the protein. Elution was performed with 500 mmol / L imidazole, and SDS PAGE separation was performed for detection. The concentration of the recombinant smooth muscle myosin with a fusion histidine tag was 0.5 mg / mL, which could be used for animal immunization and antibody screening and identification.

[0029] Example 2 Establishment of hybridoma cell lines

[0030] I. Immunization

[0031] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week old female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection of 6 points per mouse at a dose of 20 μg per mouse. The mice were boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. Seven days after the third boost, the serum of the mice was tested for the titer of polyclonal antibodies against the immunogen by indirect ELISA (wavelength 450 nm). The mouse with the highest titer was given a boost by tail vein injection of the antigen mixed with normal saline at a dose of 20 μg per mouse.

[0032] II. Cell fusion

[0033] The spleen cell suspension of the immunized mice was prepared aseptically and mixed with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the centrifuge tube was placed in a 37°C water bath. Within 1 min, 1 mL of PEG1500 (Roche) was slowly added while stirring the cells. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10 mL of serum (PAA) was carefully added to the cells, which were then blown up, and 5 mL of mixed 10x HAT (Sigma) was added, mixed well. Then, 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed well, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in a 37°C 5% CO2 incubator.

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

[0035] Seven days after fusion, the size and density of the cell colonies were moderate. Under a dissecting microscope, round, solid, and large colonies were sucked into 96-well culture plates containing prepared medium and cultured in a 37°C 5% CO2 incubator. After 3 days, the cells occupied about 2 / 3 of the bottom area, and 100 μL of supernatant was taken for ELISA screening with the immunogen and synthetic polypeptide, respectively. The positive colonies were completely changed, and 200 μL of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening was performed, and the positive colonies were transferred to 24-well plates containing prepared medium (containing feeder cells and HT) for culture. Five days later, 100 μL of supernatant was taken for the third ELISA screening, and the positive colonies were successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

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

[0037] I. Ascites preparation

[0038] Logarithmic growth phase cells were washed with serum-free medium and suspended, and counted to be about 5 x 106cells / mL. 1 mL of the suspended cells was injected intraperitoneally into a mouse previously sensitized with paraffin oil. Ascites was collected 7 days later. The collected ascites was centrifuged at 4000 rpm for 10 min at 4°C. The middle ascites was carefully sucked out and collected in a centrifuge tube, which was stored at 4°C or -20°C. 5

[0039] II. Purification of monoclonal antibody

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

[0041] Example 4 Identification of characteristics of monoclonal antibody

[0042] I. Subclass identification

[0043] Sheep anti-mouse IgG (Beijing Zhongshanjinqiu Biotechnology Co., Ltd.) was diluted to 0.5 μg / mL with 100 mM PBS (pH 7.4), 100 μL was added to each well, and incubated at 4°C overnight. The liquid was poured out, and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200 μL of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 0.1 mL of HRP-labeled sheep anti-mouse (κ, λ) antibody diluted 1:1000 or HRP-labeled sheep anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody diluted 1:2000 (Southern Biotech) was added to appropriate wells, respectively, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. Color development was performed by adding 50 μL of citric acid buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 to each well, and the OD value at 405 nm was determined within 10-20 min.

[0044] The results show that the monoclonal antibody of the application is an IgG2b type mouse monoclonal antibody.

[0045] II. Determination of affinity constant

[0046] ​Coat the smooth muscle myosin recombinant protein prepared in Example 3 at a coating concentration of 2 μg / mL, 100 μL / well, at 4°C overnight, and wash 3 times with PBS-T. Add 200 μL blocking solution to each well, and block at 37°C for 2 h, and wash 3 times with PBS-T. Dilute the monoclonal antibody purified in Example 3 by gradient dilution by 2 times starting from 1:200, and leave one well as a blank control, and incubate at 37°C for 1 h, and wash 3 times with PBS-T. Dilute HRP-labeled goat anti-mouse secondary antibody at 1:20,000, and add 100 μL to each well, and incubate at 37°C for 1 h, and wash 3 times with PBS-T. Add 100 μL of citric acid-phosphoric acid buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 to each well, and develop for 10 min, and add 50 μL of 0.5 M sulfuric acid solution to stop the reaction. Measure the absorbance at a wavelength of 450 nm using an enzyme-labeled instrument. Plot the OD value against the dilution factor of the antibody, and find the dilution factor A corresponding to half of the maximum binding OD value. Calculate the affinity constant of the antibody to be 1.92 x 10 9 .

[0047]

[0048] III. Specificity of monoclonal antibody reaction and application effect

[0049] Take the smooth muscle myosin recombinant protein prepared in Example 1, and detect the recognition specificity of the monoclonal antibody of the application by immunoblotting, and perform 12% polyacrylamide gel electrophoresis. Transfer the gel protein band to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to a conventional method. Place the membrane in a TBS-T blocking solution containing 5% nonfat milk at 4°C overnight. Add the smooth muscle myosin protein monoclonal antibody purified in Example 3 (diluted at 1:1000) and incubate at 4°C overnight. After washing the membrane with TBS-T, add goat anti-mouse secondary antibody (Beijing Zhongshen Jinqiao Biotechnology Co., Ltd.) diluted at 1:5000, and incubate at room temperature for 1 h. Wash the membrane again with TBST, add ECL hypersensitive developing solution (Beijing Puli Lei Gene Technology Co., Ltd.), and collect chemiluminescence image data using a ChemiDoc MP multicolor fluorescence imaging system (Bio-Rad).

[0050] Example 5: Determination of variable region sequence of antibody

[0051] Take fresh hybridoma cells in culture, and take the supernatant to verify the antigen binding property, and confirm that the cell strain used for cloning can indeed secrete the required antibody. After the result is confirmed, centrifuge to collect 10 6The above hybridoma cells. Trizol method was used to extract total RNA from hybridoma cells. 9 μL of total RNA, 2.5 μL of oligo (dT) 12-18 primer (10 mM), and 5 μL of dNTPs were mixed uniformly, and then incubated at 70 °C for 5 min and placed on ice for 5 min, or denatured according to the reverse transcriptase used. Then 5 μL of RT buffer (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 h. The reaction was terminated by incubation at 70 °C for 15 min, and the obtained cDNA was stored at -20 °C. The obtained first-strand cDNA was subjected to PCR amplification. In a 50 μL reaction system, 25 pmol of primers were added, and the sequences of the primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the primer sequences in the book "Recombinant Antibodies" (Science Press, published in 2005) edited by Shen Bei-fen.

[0052] The remaining dNTPs and buffers were added according to the conventional method, and finally 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set as 94 °C for 40 s, 52 °C for 40 s, and 72 °C for 40 s, and 20 to 25 cycles were performed, and finally 72 °C was extended for 3 min. The product can be stored at 4 °C or directly electrophoresed. 20 μL of the PCR product was subjected to electrophoresis analysis, separated on a 1.5% agarose gel, and the length of the light chain (κ light chain) was between 320-340 bp and the length of the heavy chain was between 340-370 bp. When specific products in this region were obtained, the gel was cut and recovered, cloned into a T vector or an expression vector, and sequenced.

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

[0054] I. Chip preparation process

[0055] Each sample was first subjected to HE section staining to determine the tumor site. A full-automatic tissue chip instrument of 3DHISTECH Company was used to make the tissue chip. The prepared tissue chip wax block was placed into a wax block making mold, and then placed into a 68 °C oven for 10 min, so that the wax of the tissue chip and the wax of the receptor wax block were integrated, and then the mold was gently taken out of the oven, and the semi-melted paraffin was cooled at room temperature for about 30 min, and then placed into a -20 °C refrigerator for freezing for 6 min, and then the tissue chip wax block was taken out of the mold, and sectioned or stored in a 4 °C refrigerator for standby use. After trimming, continuous sections were cut with a thickness of 3 μm, and then the continuous sections were floated in 40% alcohol to naturally expand, and then the separated sections were transferred to warm water at 50 °C for 30 s, and then the sections were mounted on glass slides treated with polylysine, and then the prepared tissue chip was placed into a 68 °C oven for 2 h for sectioning, and then taken out, cooled at room temperature, and stored in a -4 °C refrigerator.

[0056] II. IHC staining and analysis

[0057] Routine dewaxing of xylene 3 times, 6 minutes each, 100%, 100%, 95%, 85% gradient ethanol hydration, 3 minutes each, last tap water rinse. Antigen retrieval was performed, then the sections were placed in a wet box and rinsed in PBS 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, rinsed in PBS 3 x 3 minutes. The sections were spun down and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody to dilute the antibody ratio) and incubated at room temperature (25°C) for 1 hour, rinsed in PBS 3 x 3 minutes, the secondary antibody was added and incubated at room temperature for 15-30 minutes, rinsed in PBS 3 x 3 minutes, the PBS was spun off and fresh DAB developing solution was added and developed for 3-10 minutes. Hematoxylin counterstained for 25 seconds and returned to blue for 30 seconds in PBS. Dehydrated in alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes) in turn, finally xylene transparent for 3 minutes, neutral resin mounting.

[0058] The results of immunohistochemical staining are divided into: positive and negative. Positive expression must be at the site of cell and tissue specific antigen to be considered positive. In the case of clear distribution of tissue staining 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, marked as "-".

[0063] III. Data Statistics

[0064] 1. Tumor tissue chip detection results:

[0065] The breast cancer of 62 cases was detected synchronously by the antibody smooth muscle myosin (15C1D4) and the commercially available antibody smooth muscle myosin antibody (SMMS-1), and the detection results were compared.

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

[0067]

[0068] The results show that the prepared anti-smooth muscle myosin monoclonal antibody has accurate staining positioning, clear staining and no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate is equivalent to that of the commercial antibody, but the positive intensity of 2 cases is higher than that of the commercial antibody. It is indicated that the prepared smooth muscle myosin has higher sensitivity, and effectively avoids false negative results.

[0069] Figure 1 A comparison chart of breast cancer immunohistochemical staining results (left: the antibody prepared in the application, right: the commercial antibody).

[0070] 2. Normal tissue chip test results:

[0071] The normal tissue chip includes 30 normal tissue samples, and the normal tissue samples 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, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0072] The prepared antibody (15C1D4) and the commercial antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, which indicates that the specificity of the prepared antibody in the normal tissue is equivalent to that of the commercial antibody. Figure 2 A comparison chart of intestinal surface epithelial immunohistochemical staining results ((left: the antibody prepared in the application, right: the commercial antibody).

[0073] Finally, it should be noted that although the above-mentioned embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solutions obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are all included in the patent protection scope of the present application.

Claims

1. A monoclonal antibody against smooth muscle myosin, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the light chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. A smooth muscle myosin immunoassay reagent, characterized in that, The immunoassay reagent contains the anti-smooth muscle myosin monoclonal antibody as described in claim 1 as its active ingredient.

3. The immunoassay reagent according to claim 2, characterized in that, The immunoassays include immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

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