Monoclonal antibody against myogenin protein and preparation method and application thereof

Abstract

The application relates to a kind of monoclonal antibody that can identify human Myogenin antigen, its preparation method and its use in immunodetection.The above technical solution selects the amino acid of 1-224 of Myogenin protein as antigen peptide, is codon optimized, becomes the gene fragment suitable for expressing in escherichia coli BL21, and finally the recombinant protein contains Myogenin protein fragment and histidine protein label.The recombinant protein is immunized to mouse, is fused to cell, is screened and subcloned, obtains the mouse hybridoma cell strain of secreting anti-Myogenin protein monoclonal antibody and the anti-Myogenin protein monoclonal antibody secreted by the cell strain.The antibody obtained in the scheme has high specificity, sensitivity, can specifically recognize the cell expressing Myogenin protein, 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-Myogenin protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Myogenin, also known as MyoD1 or Myf4, is a member of the myogenic regulatory factor (MRFs) family, which plays a key role in skeletal muscle development and differentiation. It was first isolated from a myoblast-specific cDNA library by Wright et al. in 1988 using a method of sequence homology screening. Myogenin is a transcription factor that promotes myoblast (i.e., myogenic cell) fusion into myotubes and further differentiation into mature muscle fibers by regulating the expression of muscle-specific genes. Studies by Hasty and Weintraub et al. have shown that Myogenin plays a central role in the MyoD family and is particularly important in the later stages of muscle development, serving as a key regulatory point in the process of muscle cell differentiation.

[0003] Myogenin is located on chromosome 1 and has a relative molecular mass of about 25 kDa. The exact number of amino acids varies depending on the species and protein subtype, but is generally close to 200 amino acids. Myogenin is one of the important immunohistochemical markers in the diagnosis of rhabdomyosarcoma (RMS). In the diagnosis of RMS, positive expression of Myogenin is often closely related to the diagnosis of RMS. In combination with other immunohistochemical markers such as Desmin, Myoglobin, etc., the accuracy and reliability of RMS diagnosis can be further improved.

[0004] Although Myogenin is mainly associated with muscle tissue, abnormal expression of Myogenin has also been found in some non-muscle-derived tumors, such as certain subtypes of lung cancer or breast cancer. However, these findings are relatively few, and the specific mechanisms need further study. SUMMARY

[0005] The present application provides an anti-Myogenin protein 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.

[0006] Further, the monoclonal antibody is a murine monoclonal antibody with clone number 55B9F5.

[0007] The present application also provides a preparation method of an anti-Myogenin protein monoclonal antibody, wherein the antigen for immunizing mice is a recombinant protein expressed by E. coli.

[0008] Further, the recombinant protein comprises a Myogenin protein fragment and a HIS protein tag.

[0009] Further, the Myogenin protein fragment is a fragment of 1-224 sites, which is an amino acid sequence shown in SEQ ID NO. 3.

[0010] Further, the plasmid vector selected in the recombinant process is pET30a.

[0011] The inventor also provides a Myogenin protein immunodetection reagent, which contains the above-mentioned anti-Myogenin protein monoclonal antibody as an effective component.

[0012] Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

[0013] Differing from the prior art, the beneficial technical effects of the present application are that the above-mentioned technical solution selects the amino acid of 1-224 sites of Myogenin protein as an antigen peptide, which is codon-optimized to become a gene fragment suitable for expression in Escherichia coli BL21(DE3), and finally a recombinant protein comprising a Myogenin protein 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 55B9F5 secreting anti-Myogenin protein monoclonal antibody is obtained, as well as the anti-Myogenin protein monoclonal antibody secreted by the cell strain. The antibody obtained by the present solution has high specificity and sensitivity, and can specifically recognize cells expressing Myogenin protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a comparison chart of immunohistochemical staining results of one rhabdomyosarcoma; the left is the antibody of the present application, and the right is a commercially available antibody.

[0015] Figure 2 It is a comparison chart of immunohistochemical staining results of skeletal muscle; the left is the antibody of the present application, and the right is a commercially available antibody. DETAILED DESCRIPTION

[0016] In order to explain the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects that can be achieved 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.

[0017] The term "embodiment" is mentioned in this document means that the specific features, structures or properties 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.

[0018] 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.

[0019] 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.

[0020] In this application, such as "first" and "second", the terms are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0021] 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.

[0022] As the same understanding 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, in the description of the embodiments of the present application, the meaning of "multiple" 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.

[0023] Preparation of recombinant Myogenin protein fragment of Example 1

[0024] I. Gene optimization and synthesis

[0025] Myogenin according to the protein sequence of accession number NP_002470.2 in NCBI database, the protein fragment of 1-224 amino acids was selected and 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.

[0026] 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 cells of E. coli 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.

[0027] Different antigens may be selected for immunization to prepare 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 Myogenin 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 1-224 of Myogenin were subjected to codon optimization, and the molecular weight was about 25-35 kDa. The Myogenin protein was obtained by sequence optimization design using prokaryotic expression gene sequence. The recombinant immunogen is composed of Myogenin protein fragments with antigenicity and protein tags for purification of recombinant proteins. The protein tag is HIS.

[0028] II. Protein expression and purification

[0029] The single colony culture overnight bacteria were transferred to 100 mL 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 collecting the bacteria, ultrasonic disruption was performed. 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 and detection were performed. The concentration of the recombinant Myogenin protein with a fusion histidine tag was 0.5 mg / mL, which could be used for animal immunization and antibody screening and identification.

[0030] Example 2 Establishment of hybridoma cell lines

[0031] I. Immunization

[0032] 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 anti-antigen polyclonal antibodies 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.

[0033] II. Cell fusion

[0034] 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 and the cells were stirred. 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 blown up, and 5 mL of 10x HAT (Sigma) mixed with thymocytes 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.

[0035] III. Cloning and ELISA screening of positive hybridoma cells

[0036] Seven days after fusion, the size and density of the cell colonies were moderate, and under a dissecting microscope, round, solid, and large colonies were sucked into 96-well culture plates containing 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 clones 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 clones were transferred to 24-well plates containing 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 clones were successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

[0037] I. Preparation of ascites

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

[0039] II. Purification of the monoclonal antibody

[0040] The antibody was purified from the 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 the characteristics of the monoclonal antibody

[0042] I. Subclass identification

[0043] Sheep anti-mouse IgG (Beijing Zhongshanjinqiao 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 or 1:2000 diluted HRP-labeled sheep anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) was added to the appropriate wells, respectively, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 50 μL of citric acid buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 was added to each well for color development, and the OD value at 405 nm was determined within 10-20 min.

[0044] The results show that the monoclonal antibody of the present application is an IgG1 type mouse monoclonal antibody.

[0045] II. Determination of affinity constant

[0046] Coat Myogenin recombinant protein prepared in Example 3, coating concentration is 2 μg / mL, 100 μL / well, 4°C coating overnight, PBS-T wash 3 times. Add 200 μL blocking solution to each well, 37°C blocking for 2 hours, PBS-T wash 3 times. The monoclonal antibody purified in Example 3, start from 1:200, 2-fold gradient dilution, the last one well left blank control, 37°C incubation for 1 hour, PBS-T wash 3 times. HRP labeled goat anti-mouse secondary antibody 1:20000 dilution, 100 μL per well, 37°C incubation for 1 hour, PBS-T wash 3 times. Add 100 μL of 0.1% TMB (Sigma) and 0.03% H2O2 containing citric acid-phosphoric acid buffer to each well, color for 10 minutes, add 50 μL of 0.5M sulfuric acid solution to stop the reaction. Measure the absorbance value at 450 nm with a microplate reader. Draw the curve of OD value corresponding to the dilution of the antibody, find the dilution A corresponding to half of the maximum binding OD value, and calculate the affinity constant of the antibody as 1.92 x 10 9 .

[0047]

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

[0049] Take the Myogenin recombinant protein prepared in Example 1, and use the Western blot method to detect the recognition specificity of the monoclonal antibody of the application, and perform 12% polyacrylamide gel electrophoresis. The gel protein band is transferred to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane is placed in a TBS-T blocking solution containing 5% skim milk at 4°C overnight. Add the Myogenin protein monoclonal antibody purified in Example 3 (1:1000 dilution) and incubate at 4°C overnight. After washing the membrane with TBS-T, add goat anti-mouse secondary antibody (Beijing Zhongshanjingqiao Biotechnology Co., Ltd.) diluted 1:5000 and incubate at room temperature for 1 hour. Wash the membrane with TBST again, add ECL ultra-sensitive developing solution (Beijing Puli Lei Gene Technology Co., Ltd.), and collect the chemiluminescence image data with a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

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

[0051] Take fresh hybridoma cells in culture, and take the supernatant to verify the antigen binding property, to 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, and finally tap water rinse. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS 3x3 minutes. 3% H2O2 was added dropwise and incubated for 10 minutes, and then rinsed with PBS 3x3 minutes. The sections were spun dry, the appropriate dilution of primary antibody was added dropwise (the first dilution was designed according to the concentration of the antibody to dilute the antibody), incubated at room temperature (25°C) for 1 hour, rinsed with PBS 3x3 minutes, the secondary antibody was added dropwise and incubated at room temperature for 15-30 minutes, rinsed with PBS 3x3 minutes, the PBS was spun off, and fresh DAB developing solution was used to develop for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and PBS was returned to blue for 30 seconds. Sequential dehydration was performed according to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing for 3 minutes, and 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 antigens 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, marked as "-".

[0063] III. Data Statistics

[0064] 1. Tumor tissue chip detection results:

[0065] The antibody Myogenin (55B9F5) of the application and the commercially available antibody Myogenin (F5D) were used to synchronously detect 57 cases of rhabdomyosarcoma and compare the detection results.

[0066] The immunohistochemical results of Myogenin were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are as follows:

[0067]

[0068] The results show that the anti-Myogenin protein monoclonal antibody provided by the application has accurate staining positioning, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive rate is equivalent to that of the commercial antibody, but the positive intensity of 3 cases is higher than that of the commercial antibody. It is indicated that the antibody has higher sensitivity and effectively avoids false negative results.

[0069] Figure 1 Figure is a comparison chart of skeletal muscle immunohistochemical staining results (left is the antibody of the application, and right is 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 antibody (55B9F5) and the commercial antibody are synchronously detected on the normal tissue chip, the positive and negative detection results are consistent, and it is indicated that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody. Figure 2 Figure is a comparison chart of skeletal muscle immunohistochemical staining results (left is the antibody of the application, and right is 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 application, the patent protection scope of the application should not be limited. Any equivalent structure or equivalent flow replacement or modification based on the essential concept of the application, the content described in the specification and drawings, and the direct or indirect implementation of the technical solutions of the above embodiments in other related technical fields, are all included in the patent protection scope of the application.

Claims

1. An anti-Myogenin protein monoclonal antibody, characterized by, 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. An immunoassay reagent for Myogenin protein, characterized by comprising an antibody which recognizes the amino acid sequence of SEQ ID NO:

1. The immunodetection reagent contains the anti-Myogenin protein monoclonal antibody of claim 1 as an effective component.

3. The immunoassay test reagent of claim 2, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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