CN117700544B

CN117700544B - anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof

Info

Publication number: CN117700544B
Application number: CN202410160104.2A
Authority: CN
Inventors: 肖目张; 徐志杰; 何青春; 刘杨; 张东旭
Original assignee: Suzhou Baidao Medical Technology Co ltd; Xiangya Hospital of Central South University
Current assignee: Suzhou Baidao Medical Technology Co ltd; Xiangya Hospital of Central South University
Priority date: 2024-02-05
Filing date: 2024-02-05
Publication date: 2024-04-16
Anticipated expiration: 2044-02-05
Also published as: CN117700544A

Abstract

The invention belongs to the technical field of immunochemistry, and relates to an anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof, wherein the anti-MSH 2 recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is as follows: an amino acid sequence as shown in SEQ ID No. 4; or a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 4; the light chain variable region is: an amino acid sequence as shown in SEQ ID No. 5; or a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 5. The anti-MSH 2 recombinant rabbit monoclonal antibody provided by the invention has high affinity with MSH2 protein, and can recognize and detect the expression of MSH2 protein on cells with high specificity and high sensitivity.

Description

anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof, in particular to application in immunohistochemical detection.

Background

The Mismatch Repair system (MMR) is an important DNA Repair mechanism, a system that recognizes and repairs false insertions, deletions and mismatches of bases that may occur during DNA replication or recombination, and repairs some forms of DNA damage, preventing genetic mutations to maintain genome stability. The system consists of a series of specific DNA mismatch repair enzymes. MMR gene germ line detection is a "gold standard" for diagnosis of Lynch Syndrome. Molecular etiologies of Lynch syndrome include heterozygous pathogenic mutations on MLH1, MSH2, MSH6 or PMS2 genes in the MMR gene family, as well as EPCAM gene deletion mutations (which result in hypermethylation of the MSH2 promoter, causing silencing of the MSH2 gene). Because MMR genes are functionally inactivated, lynch syndrome patients often show MSI-H and dMMR, so MSI detection and MMR protein IHC detection can be used as a primary screening means.

In immunohistochemistry, mismatch repair proteins generally comprise four items of MSH2, MSH6, MLH1 and PMS2, and are generally applied to screening of hereditary non-polyposis intestinal cancer (HNPCC) and tumors accompanied by MSI in a combined way. Wherein MSH2 is a mismatch repair protein having a molecular weight of about 104 kD. MLH2 can form heterodimer with MSH6, and MSH2/MSH6 heterodimer is responsible for combining with mismatched base of initial DNA through conformational change and has the function of recognizing single base mismatch such as mismatch mutation, short deletion, insertion mutation and the like. Mutation of this gene leads to defective mismatch repair functions in the cell, destabilizing Microsatellites (MSI), and thus to tumor susceptibility.

Immunohistochemical (IHC) methods judge whether MMR is defective by microscopic observation of normal intracellular expression. However, the sensitivity and specificity of the existing anti-MSH 2 antibody are still to be improved, so that the evaluation in IHC staining is difficult, and the accuracy of detecting and distinguishing cancers is affected. The ELISA kit which is visible in the market at present has low detection sensitivity and weak positive signals, and is not easy to identify in IHC staining, so that the screening of a high-sensitivity and strong-specificity anti-MSH 2 antibody has very important effects on identifying and detecting the expression of MSH2 protein on cells.

Disclosure of Invention

First, the technical problem to be solved

The invention provides an anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof, and aims to solve the problems that the existing anti-MSH 2 recombinant rabbit monoclonal antibody has weak positive signals and is unfavorable for recognition in IHC (IHC) dyeing.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

in a first aspect, the invention provides an anti-MSH 2 recombinant rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region,

the heavy chain variable region is: an amino acid sequence as shown in SEQ ID No. 4; or a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 4;

the light chain variable region is: an amino acid sequence as shown in SEQ ID No. 5; or a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 5.

In a second aspect, the invention provides a nucleotide encoding said anti-MSH 2 recombinant rabbit monoclonal antibody.

Preferably, the nucleotide comprises a DNA sequence as shown in SEQ ID No.2 for encoding the heavy chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody; and a DNA sequence shown as SEQ ID No.3 is used for encoding a light chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody.

In a third aspect, the invention relates to a nucleic acid molecule comprising a nucleotide for encoding said anti-MSH 2 recombinant rabbit monoclonal antibody.

In a fourth aspect, the invention provides a biomaterial comprising a nucleic acid molecule as described above, said biomaterial being an expression vector or a recombinant plasmid.

In a fifth aspect, the invention provides a method for preparing an anti-MSH 2 recombinant rabbit monoclonal antibody, which comprises the steps of transfecting cells by adopting the expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-MSH 2 recombinant rabbit monoclonal antibody.

More preferably, the preparation method comprises the steps of:

(1) Immunization of rabbits: firstly, analyzing the MSH2 protein molecular sequence, selecting and using a proper polypeptide sequence as an immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and a secondary structure of MSH2, coupling the polypeptide sequence by KLH or OVA, and immunizing rabbits; the polypeptide is an artificially synthesized polypeptide shown in SEQ ID No. 1;

(2) Preparation of hybridoma cell lines: obtaining a positive hybridoma stable cell line capable of efficiently secreting antibodies through cell fusion, cloning and screening, and separating total RNA from the hybridoma cell line;

(3) Obtaining an antibody sequence: the nucleotide sequence SEQ ID No.2 of the antibody heavy chain variable region and the nucleotide sequence SEQ ID No.3 of the antibody light chain variable region are obtained by utilizing specific primers through a PCR amplification technology;

(4) Antibody expression and purification: cloning the nucleotide sequence into an expression vector, transiently transfecting the cultured cells by using a transfection method, collecting the supernatant after the culture, and purifying the supernatant by using Protein A to obtain the antibody with the purity of more than 95 percent.

In a sixth aspect, the anti-MSH 2 recombinant rabbit monoclonal antibody, nucleotide, nucleic acid molecule, expression vector or recombinant plasmid is applied to preparation of MSH2 protein molecule detection device. The detection device includes, but is not limited to, a kit, an antibody chip, and the like.

In a seventh aspect, the invention also provides a MSH2 assay kit comprising the anti-MSH 2 recombinant rabbit monoclonal antibody and an immunohistochemical assay reagent as described above.

Preferably, the MSH2 detection kit further includes at least one of a substrate chromogenic solution, a blocking solution, or a repair solution.

Further preferred includes: anti-MSH 2 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody (HRP enzyme-labeled secondary antibody), EDTA repair liquid, catalase blocking liquid, 3-diaminobenzidine concentrate (DAB concentrate), 3-diaminobenzidine buffer (DAB buffer), hematoxylin and blue returning liquid.

In the case of immunohistochemical detection, the detection steps comprise dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, 3-diaminobenzidine chromogenic (DAB chromogenic), counterstaining, dehydration, sealing and microscopic examination, and the like.

(III) beneficial effects

The invention provides an anti-MSH 2 recombinant rabbit monoclonal antibody, and also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-MSH 2 recombinant rabbit monoclonal antibody, a preparation method, application of the anti-MSH 2 recombinant rabbit monoclonal antibody in an MSH2 protein detection method or device and the like. The combination of the anti-MSH 2 recombinant rabbit monoclonal antibody and MSH2 protein molecules has high specificity and high sensitivity, can specifically identify and detect the expression of MSH2 protein on cells, and has positive high expression when detecting MSH2 protein, so the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, and is beneficial to obtaining accurate evaluation and detection results. The MSH2 recombinant rabbit monoclonal antibody cloned by 644G5A4 has the characteristics of good specificity, strong positive signals and the like, is easier to score in IHC staining, and is more accurate for detecting and distinguishing cancers. The method can be applied to the detection and screening fields of immunohistochemistry, indirect ELISA, immunoblotting, antibody chip preparation and the like, is favorable for obtaining more accurate detection and evaluation results, and reduces detection cost and interference of background signals.

Drawings

FIG. 1 is a graph showing the results of immunohistochemical detection of 644G5A4 anti-MSH 2 monoclonal antibody and commercial antibody prepared in the present invention in human tonsil, colon cancer and breast cancer tissues, wherein the 644G5A4 anti-MSH 2 monoclonal antibody is used at a concentration of 0.5 μg/mL; wherein a is an immunohistochemical detection result diagram of the MLH1 antibody cloned by 644G5A4 on tonsils, and b is an immunohistochemical detection result diagram of a commercially available antibody on tonsils; c is an immunohistochemical detection result diagram of the MLH1 antibody cloned by 644G5A4 in colon cancer, d is an immunohistochemical detection result diagram of a commercial antibody in colon cancer; e is a diagram of the immunohistochemical detection result of the MLH1 antibody cloned in 644G5A4 in breast cancer, and f is a diagram of the immunohistochemical detection result of the commercial antibody in breast cancer.

FIG. 2 is a statistical plot of the titer detection of 644G5A4 anti-MSH 2 monoclonal antibodies and commercially available antibodies of the invention at 8 gradient concentrations.

FIG. 3 shows the result of immunoblotting (Western blotting) detection of the 644G5A4 anti-MSH 2 monoclonal antibody as the primary antibody according to the present invention to verify the recognition capability of MSH2 protein.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted. The human tissue samples are formalin-fixed and paraffin-embedded human tissue samples, and are subjected to pathological confirmation and informed consent of patients.

The anti-MSH 2 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-MSH 2 recombinant rabbit monoclonal antibody provided by the invention is produced by eukaryotic expression of 293 cells through rabbit hybridoma fusion screening. When the anti-MSH 2 monoclonal antibody is prepared, an antigen for immunizing rabbits (New Zealand white rabbits) is a synthetic polypeptide, the amino acid sequence of the synthetic polypeptide is shown as SEQ ID No.1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After immunization, a positive hybridoma cell line capable of efficiently secreting monoclonal antibodies is obtained through cell fusion, clone screening, a molecular cloning technology is used for obtaining nucleotide sequences of heavy chain amino acid sequences and light chain amino acid sequences of the antibodies, the nucleotide sequences are constructed on eukaryotic expression vectors, the eukaryotic expression vectors are transfected into 293 cell lines through transfection reagents, cell supernatants are collected, and the cell supernatants are purified through Protein A column affinity chromatography, so that rabbit monoclonal antibodies are obtained. Immunohistochemical detection shows that the antibody can specifically recognize MSH2 protein. The anti-MSH 2 monoclonal antibody can recognize recombinant MSH2 antigen protein and MSH2 molecules on cells; the anti-MSH 2 recombinant rabbit monoclonal antibody (MSH 2 rabbit antibody) provided by the invention can be used for immunohistochemical detection, and can recognize and detect the expression of MSH2 protein on cells with high specificity and high sensitivity. The anti-MSH 2 monoclonal antibody can also be applied to an immunohistochemical pathological diagnostic agent.

Example 1

This example is the preparation and screening of anti-MSH 2 recombinant rabbit monoclonal antibodies, comprising the steps of:

(1) Antigen preparation

The polypeptide sequence of the MSH2 antigen is shown as SEQ ID NO. 1.

SEQ ID No. 1：FVNEIISRIKVTT。

The polypeptide sequence is selected by analyzing the MSH2 molecular sequence according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure of MSH2 protein molecules. The polypeptide with the sequence shown in SEQ ID NO.1 is synthesized artificially, and the synthesized polypeptide is used as an antigen for immunizing rabbits. In immunization, the polypeptide with the sequence shown in SEQ ID NO.1 is coupled through KLH or OVA and then used as an immunogen to immunize rabbits.

(2) Immunization

The polypeptide sequence (MSH 2 antigen) of SEQ ID NO.1 is respectively mixed with complete Freund's adjuvant in an equal volume and emulsified, a plurality of New Zealand white rabbits are respectively immunized by a subcutaneous multipoint injection method, and the MSH2 antigen containing the polypeptide sequence (shown as SEQ ID NO. 1) is subjected to second and third immunization by equal volume emulsification with incomplete Freund's adjuvant after two weeks of interval. Blood is taken after three immunization, and serum titer is measured by ELISA method gradient dilution; and selecting the rabbit with the highest immune antibody titer with the SEQ ID NO.1 antigen for the next cell fusion.

(3) Cell fusion

Taking rabbit spleen of rabbit with highest immune antibody titer with SEQ ID NO.1 antigen to prepare lymphocyte single cell suspension; mixing rabbit spleen lymphocytes with the myeloma cells, dripping 50% PEG1500, adding IMDM culture medium, centrifuging, removing supernatant, and adding HAT cultureThe base is gently suspended and evenly mixed, the base is split into 96-well plates after being fixed to 800mL, and the base is placed at 37 ℃ and 5 percent CO ₂ Culturing in a constant temperature incubator. After 6-9 days of fusion, the state of the fused cells in the 96-well plate is observed, and the solution is replaced by HT and is continuously placed at 37 ℃ and 5% CO ₂ Culturing in a constant temperature incubator. The myeloma cells are sp2/0 myeloma cells derived from mice in advance, and the sp2/0 myeloma cells are in the logarithmic growth phase when fused.

(4) Screening and cloning

Cloned cells were screened 7-10 days after fusion using an ELISA test with MSH2 antigen (SEQ ID NO: 1). And labeling corresponding cell strain numbers, and carrying out limiting dilution on positive hole cells until the result of ELISA measurement of the whole 96-well plate is positive. And selecting a monoclonal stable strain with high positive value to obtain a hybridoma cell strain secreting the specific monoclonal antibody, and recording the hybridoma cell strain as 644G5A4.

(5) Antibody sequencing

The screened hybridoma cell strain 644G5A4 is separated from 644G5A4 hybridoma cells according to the reagent TriZol instruction, the total RNA is reversely transcribed into cDNA according to the TIANScript first strand cDNA synthesis kit instruction, the nucleotide sequences of an antibody heavy chain variable region and an antibody light chain variable region are obtained by amplifying a specific primer (a heavy chain variable region primer, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-R GTGAGGGTGCCCGAG and a light chain variable region primer: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG), and then the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region are cloned into a eukaryotic expression vector (InvivoGen, pfuse-rchg, pfuse2-rclk 1) to prepare for cell transfection.

(6) Cell transfection and screening

293 cells to be transfected are prepared in advance, fresh culture medium is centrifugally replaced and then is respectively put into 24-well plates, 1.5ml of culture medium is added into each well according to the required quantity, and the density is 3 multiplied by 10 ⁶ And each ml.

Mixing the eukaryotic expression vector and Polyethylenimine (PEI) according to the mass ratio of 1:6, adding the mixture into prepared 293 cells, and placing the mixture at 37 ℃ and 5% CO ₂ Is cultured in a shaker. Transfected after 3-5 days of cultureELISA detection is carried out on cell supernatant and corresponding antigen to screen positive holes, then immunohistochemical detection is carried out on the cell supernatant of the positive holes, and if the immunohistochemical detection is positive, the detected antibody sequence is correct.

(7) Preparation and purification of cell-on-list antibodies

And (3) carrying out a large number of cell transfection on the expression vector with positive confirmation, continuously culturing for 3-5 days, collecting cell suspension, centrifuging, taking supernatant, and purifying by using an affinity chromatography method. Measuring the concentration of the purified monoclonal antibody, sub-packaging, and storing in a refrigerator at 4-8 ℃.

Finally, the nucleotide sequence of the heavy chain variable region of the 644G5A4 anti-MSH 2 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the nucleotide sequence of the light chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No. 3.

The specific sequence of SEQ ID No.2-3 is as follows:

SEQ ID No.2：

CTTGAGGTGCAGCTGCAGGAGTCTGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTCATAGCTGGCACTGGATCCGGCTGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATTCATTACAGTGGTGGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTGTCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGGGGTACCATTGCTTACTGGGGCCAGGGAACTCTGGTCACTGTCTCTGCA。

SEQ ID No.3：

GACATTGTGCTGACCCAGTCTCCACTCACTTTGTCGGTTTCCATTGGACAACCAGCCTCCATCTCTTGCAGGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACTGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC。

and translating the obtained base sequence into an amino acid sequence, and analyzing to obtain the 644G5A4 anti-MSH 2 recombinant rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody is shown as SEQ ID No.4, and the amino acid sequence of the light chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody is shown as SEQ ID No. 5.

The specific sequence of SEQ ID No.4-5 is as follows:

SEQ ID No.4：

LEVQLQESGPDLVKPSQSLSLTCTVTGYSITSGHSWHWIRLFPGNKLEWMGYIHYSGGTNYNPSLKSRISVTRDTSKNQFFLQLNSVTTEDTATYYCARGGTIAYWGQGTLVTVSA。

SEQ ID No.5:

DIVLTQSPLTLSVSIGQPASISCRSSQSLLDSDGKTYLNWLLLRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK。

example 2 this example illustrates immunohistochemical detection of anti-MSH 2 recombinant rabbit monoclonal antibody as primary antibody by the following method:

and (3) baking the human tonsil, colon cancer and breast cancer tissue slices which are embedded by formalin-fixed paraffin for 1-2 hours in a constant temperature box, and preserving for later use. Paraffin sections were first dewaxed in fresh xylene and soaked 2 times for 10min each. The slices are soaked in absolute ethyl alcohol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes to be hydrated, and then purified water is washed for 2 times for 3 minutes each time. Repairing the slice by using a high-temperature thermal repairing method for 3min (if an automatic repairing instrument is used, the slice can be repaired at a high temperature of 98 ℃ for 20 min), naturally cooling the slice to room temperature, and then looping the tissue to be detected by using an immunohistochemical pen, and flushing with purified water for 2 times for 3min each time. Proper amount of endogenous peroxidase blocking agent is dripped to completely cover the tissue, after incubation for 10min at room temperature, purified water is washed for 2 times, each for 3min, and PBST is washed once. Then a primary antibody incubation is performed: 100. Mu.L of 0.5. Mu.g/mL 644G5A4 anti-MSH 2 recombinant rabbit monoclonal antibody was added to completely cover the tissue, and the tissue was incubated in an incubator at 37℃for 1h with 3 washes of PBST for 5min each. And then secondary antibody incubation: the secondary antibody incubation was performed according to the instructions of the DAB staining solution kit of the secondary antibody staining system used, and after incubation, the PBST was washed 3 times, 5min each time, with purified water 1 time. Preparing DAB color development liquid according to the instruction of the DAB color development liquid kit, dripping a proper amount of prepared DAB color development liquid until the DAB color development liquid completely covers tissues, stopping dyeing when the color is not deepened, and flushing with purified water for 3 times. Counterstaining the sections according to hematoxylin manufacturer instructions and advice, PBST or tap water washing to return to blue. Sequentially soaking 70%,85%,95%,100% gradient alcohol and 3min each time; 2 times the xylene is transparent, 5min each time. Finally, the samples were encapsulated with neutral gum.

FIG. 1 is a graph showing the immunohistochemical detection results of the 644G5A4 anti-MSH 2 monoclonal antibody and the commercial antibody prepared by the invention in human tonsil, colon cancer and breast cancer tissues, and the results of FIG. 1 show that MSH2 protein is specifically stained in human tonsil, colon cancer and breast cancer tissues, and the scoring in IHC staining is easier, and the detection and differentiation of cancers are more accurate. The MSH2 recombinant rabbit monoclonal antibody cloned by 644G5A4 can be used for immunohistochemical detection and immunohistochemical pathological diagnosis agents due to the characteristics of good specificity, strong positive signals and the like.

Example 3

This example is an assay for 644G5A4 anti-MSH 2 recombinant rabbit monoclonal antibody affinity, as follows:

(1) The labeled MSH2 polypeptide (SEQ ID No. 1) was removed at 4℃and returned to room temperature. And diluted to a concentration of 1. Mu.g/ml, added to a 96-well microplate at 100. Mu.L/well and incubated overnight at 4℃followed by blocking overnight at 4℃with 2% BSA. Meanwhile, the MSH2 recombinant rabbit monoclonal antibody cloned by 644G5A4 is diluted to an initial concentration of 0.5 mug/mL, and is subjected to 2-time gradient dilution in sequence, and 8 concentration gradients are set for comparison.

(2) And (2) adding the diluted anti-MSH 2 recombinant rabbit monoclonal antibody to the 96-well ELISA plate containing the polypeptide obtained in the step (1) according to 100 mu L/well, covering a sealing plate film, and incubating for 1h at a constant temperature of 37 ℃ to balance the reaction. And taking out the ELISA plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and drying the water by beating.

(3) The HRP-labeled goat anti-rabbit IgG was diluted according to the instructions for secondary antibody use, added to the elisa plate at 100 μl/well and incubated at 37 ℃ for 1h to equilibrate the reaction. And taking out the ELISA plate after the reaction is finished, discarding the liquid in the ELISA plate, flushing with purified water for 5-6 times, and drying the water by beating.

(4) TMB color development liquid is added into the enzyme label plate with the water content being beaten according to 100 mu L/hole, and the reaction is carried out for 5 to 7 minutes at room temperature. After completion of the reaction, 50. Mu.L/well of the mixture was addedIn 2M H ₂ SO ₄ The color development was terminated.

(5) OD values were read at 450nm on a microplate reader, data were collated, and the analysis results are shown in fig. 2 below.

FIG. 2 is a statistical plot of the titer detection of 644G5A4 anti-MSH 2 monoclonal antibodies and commercially available antibodies of the invention at 8 gradient concentrations. The results show that in 8 concentration gradient tests, the anti-MSH 2 recombinant rabbit monoclonal antibody cloned by 644G5A4 has strong affinity and high sensitivity to MSH2 protein molecules, when the antibody concentration is 3.90625ng/mL, the OD value of the antibody is doubled, the commercial antibody is doubled, the antibody can still reach higher OD value under the condition of lower antibody concentration, and the experiment and detection cost can be saved.

Example 4

This example illustrates immunoblotting (Western blotting) detection of anti-MSH 2 recombinant rabbit monoclonal antibody 644G5A4 as primary antibody by the following method:

(1) Selecting a PVDF membrane of 293 cells and Hela cell lysate for activation, activating methanol for 1min, washing the membrane with pure water for 2 times, and washing with TBST for 3 times; closing: placing the membrane in a blocking solution prepared from 5% BSA, and mixing and shaking for 2 hours at room temperature;

(2) Incubation resistance: diluting 644G5A4 antibody to a concentration of 0.5 mug/mL, putting the sealed membrane into the corresponding diluted antibody, and incubating at 4 ℃ with shaking overnight; taking out the membrane, putting the membrane into TBST liquid for washing for 3 times, washing for 5min each time in the former two times, and washing for 10min in the last time;

(3) Secondary antibody incubation: diluting HRP-anti-rabbit IgG with FG liquid at a ratio of 1:5000, adding membrane strips after uniformly mixing, and shaking for 1h at room temperature; taking out the membrane strip, putting the membrane strip into TBST liquid for washing for 4 times, washing for 5min each time in the first three times, and washing for 8min in the last time;

(4) A substrate: mixing equal amount of luminol/enhancing agent diluted 5 times with pure water and hydrogen peroxide solution in the same container, adding membrane strip, and incubating for 2min;

(5) Exposure: the negative film is placed in a cassette, and exposure is carried out on the X-ray film for different time periods according to the fluorescence intensity; then developing for 1min, cleaning, fixing for 1min, and finally cleaning and airing; the results are shown in FIG. 3. Wherein 293 represents a human embryonic kidney cell lysate, hela represents a human cervical cancer cell lysate; the stripe sizes are all: 105kDa.

FIG. 3 shows the result of immunoblotting (Western blotting) detection of the 644G5A4 anti-MSH 2 monoclonal antibody as the primary antibody according to the present invention to verify the recognition capability of MSH2 protein. As can be seen from the results of FIG. 3, in the lanes, the 644G5A4 anti-MSH 2 recombinant rabbit monoclonal antibody can specifically recognize MSH2 proteins in 293 cells and Hela cells, and the molecular weight is about 105kDa, which indicates that the 644G5A4 cloned MSH2 recombinant rabbit monoclonal antibody of the present invention can specifically recognize MSH2 proteins.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An anti-MSH 2 recombinant rabbit monoclonal antibody, characterized in that the anti-MSH 2 recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region,

the heavy chain variable region is: an amino acid sequence as shown in SEQ ID No. 4;

the light chain variable region is: an amino acid sequence as shown in SEQ ID No. 5.

2. A nucleotide encoding the anti-MSH 2 recombinant rabbit monoclonal antibody of claim 1.

3. The nucleotide according to claim 2, characterized in that it comprises: a DNA sequence as shown in SEQ ID No.2 for encoding the heavy chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody, and a DNA sequence as shown in SEQ ID No.3 for encoding the light chain variable region of the anti-MSH 2 recombinant rabbit monoclonal antibody.

4. A nucleic acid molecule comprising a nucleotide according to claim 2 or 3.

5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is an expression vector or a recombinant plasmid.

6. The preparation method of the anti-MSH 2 recombinant rabbit monoclonal antibody is characterized in that the expression vector of claim 5 is adopted to transfect cells, the cells are continuously cultured after transfection, and cell supernatant is collected and purified to obtain the anti-MSH 2 recombinant rabbit monoclonal antibody.

7. Use of an anti-MSH 2 recombinant rabbit monoclonal antibody according to claim 1, a nucleotide according to claim 2 or 3, a nucleic acid molecule according to claim 4, a biological material according to claim 5 for the preparation of an MSH2 detection device.

8. An MSH2 assay kit comprising an anti-MSH 2 recombinant rabbit monoclonal antibody of claim 1 and an immunohistochemical detection reagent.

9. The MSH2 assay kit of claim 8, further comprising at least one of a substrate chromogenic solution, a blocking solution, or a repair solution.