CN117700543B - anti-MLH 1 recombinant rabbit monoclonal antibody and application thereof - Google Patents
anti-MLH 1 recombinant rabbit monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to an anti-MLH 1 recombinant rabbit monoclonal antibody and application thereof, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5. The anti-MLH 1 recombinant rabbit monoclonal antibody provided by the invention has high affinity with MLH1 protein, can identify and detect the expression of MLH1 protein on tumor cells or immune cells with high specificity and high sensitivity, can be applied to the detection and screening fields of immunohistochemistry, indirect enzyme-linked immunosorbent assay, immunoblotting, antibody chip preparation and the like, is beneficial to obtaining more accurate detection and evaluation results, and reduces detection cost and interference of background signals.
Description
Technical Field
The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-MLH 1 recombinant rabbit monoclonal antibody and application thereof, in particular to application in immunohistochemical detection.
Background
MLH1 is a mismatch repair protein with a molecular weight of about 85kD, and is one of the members of the mismatch repair family. The mismatch repair system (MMR) is a form of the body's DNA repair mechanism whose main function is to correct base mismatches, preventing genetic mutations to maintain genomic stability. MLH1 can form heterodimers with PMS2 and is responsible for excision at mismatch sites and synthesis of corrected DNA strands, in particular coordinating the binding of proteins that have the function of repairing DNA errors occurring during cell division. Mutation of this gene leads to defective mismatch repair functions in the cell, destabilizing Microsatellites (MSI), and thus to tumor susceptibility. In immunohistochemistry, mismatch repair proteins generally comprise four items of MLH1, PMS2, MSH2 and MSH6, and are generally applied to screening of hereditary non-polyposis intestinal cancer (HNPCC) and tumors accompanied by MSI in a combined way.
The Immunohistochemical (IHC) method can detect MMR related proteins (MLH 1, MSH2, MSH6, PMS 2) and judge whether MMR has defects by observing whether the MMR is normally expressed in cells or not through a microscope. However, the sensitivity and specificity of the existing anti-MLH 1 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.
Therefore, the screening of a strain of highly sensitive and highly specific anti-MLH 1 antibody has a very important role in identifying and detecting the expression of MLH1 protein on cells.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the invention provides an anti-MLH 1 recombinant rabbit monoclonal antibody which has wide application and can accurately identify MLH1 expression, and through immunohistochemical detection of various different tissues, the antibody can well detect the expression of MLH1 protein on tumor cells, and can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blotting), antibody chip preparation and the like. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-MLH 1 recombinant rabbit monoclonal antibody, a preparation method, application of the anti-MLH 1 recombinant rabbit monoclonal antibody in an MLH1 protein detection method or device and the like.
(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 present invention provides an anti-MLH 1 recombinant rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of said heavy chain variable region being as shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.
The anti-MLH 1 recombinant rabbit monoclonal antibody (MLH 1 rabbit antibody) can be used for immunohistochemical detection, and can identify and detect the expression of MLH1 protein on tumor cells or immune cells with high specificity and high sensitivity.
The anti-MLH 1 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-MLH 1 recombinant rabbit monoclonal antibody provided by the invention is produced by eukaryotic expression of 293 cells through rabbit hybridoma fusion screening. In the preparation of the anti-MLH 1 monoclonal antibody, an antigen for immunizing rabbits (New Zealand white rabbits) is a synthetic polypeptide, the amino acid sequence of which is shown as SEQ ID No.1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After the rabbit is immunized, a positive hybridoma cell line capable of secreting monoclonal antibodies with high efficiency is obtained through cell fusion and 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 a eukaryotic expression vector, the eukaryotic expression vector is transfected into a 293 cell line through a transfection reagent, cell supernatants are collected, and the cell supernatants are purified through protein A column affinity chromatography, so that the rabbit monoclonal antibodies are obtained. Immunohistochemical detection shows that the antibody can specifically recognize MLH1 protein.
The anti-MLH 1 monoclonal antibody can recognize the recombinant MLH1 antigen protein and MLH1 molecules on tumor cells and immune cells; the anti-MLH 1 monoclonal antibodies may also be used in immunohistochemical pathological diagnostic agents.
In a second aspect, the present invention provides a coding gene for encoding the above anti-MLH 1 recombinant rabbit monoclonal antibody.
Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2 for coding the heavy chain variable region of the anti-MLH 1 recombinant rabbit monoclonal antibody; and a DNA sequence shown as SEQ ID No.3 for encoding a light chain variable region of the anti-MLH 1 recombinant rabbit monoclonal antibody.
In a third aspect, the present invention relates to a nucleic acid molecule comprising a coding gene for said anti-MLH 1 recombinant rabbit monoclonal antibody.
In a fourth aspect, the invention provides an expression vector or recombinant plasmid comprising a nucleic acid molecule as described above.
In a fifth aspect, the present invention provides a method for preparing an anti-MLH 1 recombinant rabbit monoclonal antibody, transfecting cells with the above expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-MLH 1 recombinant rabbit monoclonal antibody.
More preferably, the preparation method comprises the steps of:
(1) Immunization of rabbits: firstly, analyzing the MLH1 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 MLH1, and immunizing rabbits by using the immunogen after hemocyanin coupling (KLH) or Ovalbumin (OVA) coupling; the polypeptide is an artificially synthesized polypeptide shown in SEQ ID No. 1;
(2) Preparation of hybridoma cell lines: taking lymphocytes of an immunized rabbit, performing cell fusion, cloning and screening to obtain a positive hybridoma stable cell line capable of efficiently secreting antibodies, and separating total RNA from the hybridoma cell line;
(3) Obtaining an antibody sequence: reverse transcription of total RNA into cDNA, and PCR amplification to obtain the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region;
(4) Antibody expression and purification: cloning the nucleotide sequence into an expression vector, transiently transfecting the cultured cells by using a transfection method, collecting 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-MLH 1 recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid is applied to preparation of an MLH1 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 an MLH1 detection kit comprising an anti-MLH 1 recombinant rabbit monoclonal antibody as described above and an immunohistochemical detection reagent.
Preferably, the MLH1 detection kit comprises: anti-MLH 1 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody (HRP enzyme-labeled secondary antibody), ethylenediamine tetraacetic acid (EDTA) repair solution, catalase blocking solution, 3-Diaminobenzidine (DAB) concentrated solution, 3-Diaminobenzidine (DAB) buffer solution, hematoxylin and bluing solution.
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 (DAB) color development, counterstaining, dehydration, sealing and microscopic examination, and the like.
(III) beneficial effects
The anti-MLH 1 recombinant rabbit monoclonal antibody provided by the invention has high specificity and high sensitivity in combination with MLH1 protein molecules, can specifically identify and detect the expression of MLH1 protein on cells, and has positive high expression in the detection of MLH1 protein, so that the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, and is beneficial to obtaining accurate assessment and detection results. The 521I4D5 cloned MLH1 recombinant rabbit monoclonal antibody 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.
Drawings
FIG. 1 is a graph showing the results of immunohistochemical detection of 521I4D5 anti-MLH 1 monoclonal antibody and commercial antibody prepared in the present invention on tonsil, colon cancer, MLH 1-deleted colon cancer tissue, 521I4D5 anti-MLH 1 monoclonal antibody used at a concentration of 1. Mu.g/mL; wherein a is an immunohistochemical detection result diagram of an MLH1 antibody cloned by 521I4D5 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 an MLH1 antibody cloned by 521I4D5 in colon cancer, D is an immunohistochemical detection result diagram of a commercial antibody in colon cancer; e is an immunohistochemical detection result diagram of an MLH1 antibody cloned by 521I4D5 in the colon cancer tissue with MLH1 deletion, and f is an immunohistochemical detection result diagram of a commercially available antibody in the colon cancer tissue with MLH1 deletion.
FIG. 2 is a statistical plot of potency detection of 521I4D5 anti-MLH 1 monoclonal antibodies and commercially available antibodies of the invention at 8 gradient concentrations.
FIG. 3 shows the result of immunoblotting (Western blotting) assay of 521I4D5 anti-MLH 1 monoclonal antibody of the present invention as a primary antibody to verify the recognition ability of MLH1 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.
Example 1
This example is the preparation and screening of anti-MLH 1 recombinant rabbit monoclonal antibodies, comprising the steps of:
(1) Antigen preparation
The specific sequence of MLH1 antigen is shown in SEQ ID NO. 1.
SEQ ID No. 1：QLANLPDLYKVFER。
The polypeptide sequence is selected by analyzing the MLH1 molecular sequence according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure of the MLH1 protein molecule. 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 and then used as an immunogen to immunize rabbits.
(2) Immunization
The polypeptide sequence (MLH 1 antigen) of SEQ ID NO.1 is respectively mixed and emulsified with complete Freund's adjuvant (volume ratio 1:1), a plurality of New Zealand white rabbits are respectively immunized by subcutaneous injection, and the MLH1 antigen containing the polypeptide shown in SEQ ID NO.1 is emulsified with incomplete Freund's adjuvant (volume ratio 1:1) for the second and third immunization after two weeks. 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
The sp2/0 myeloma cells derived from the mice were prepared in advance, and the sp2/0 myeloma cells were allowed to be in the logarithmic growth phase at the time of fusion. Taking spleen of immunized rabbit to prepare lymphocyte single cell suspension; mixing rabbit spleen lymphocytes with myeloma cells, dripping 50% PEG1500, adding IMDM culture medium, centrifuging, removing supernatant, adding HAT culture medium, suspending gently, mixing, fixing volume to 800mL, packaging in 96-well plate, placing at 37deg.C, and 5% 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.
(4) Screening and cloning
Cloned cells were screened 7-10 days after fusion using an ELISA test with MLH1 antigen (SEQ ID NO: 1). And labeling corresponding cell strain numbers, and carrying out limiting dilution on positive hole cells until the result of the whole 96-well plate is positive by an enzyme-linked immunosorbent assay (ELISA). 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 521I4D5.
(5) Antibody sequencing is carried out on the hybridoma cell strains which are screened
Total RNA was isolated from 521I4D5 hybridoma cells according to the instructions of the TriZol RNA extraction reagent, reverse transcribed into cDNA according to the instructions of the TIANScript first strand cDNA synthesis kit, amplified using specific primers (heavy chain variable region primer, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-R GTGAGGGTGCCCGAG; light chain variable region primer: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG) to obtain nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region, and then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk 1) in preparation 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 Polyethyleneimine (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. After 3-5 days of culture, the transfected cell supernatant and the corresponding antigen are subjected to enzyme-linked immunosorbent assay (ELISA) detection to screen positive holes, and then the cell supernatant of the positive holes is continuously subjected to immunohistochemical detection, 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 heavy chain variable region nucleotide sequence of the 521I4D5 anti-MLH 1 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the light chain variable region nucleotide sequence of the anti-MLH 1 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：
CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACGGATTACTTGATAGAGTGGGTAAAGCAGTGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTAATACTGACTACAATGAGAAGTTCAGGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCATCTCACCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTACAAGATGGGTGAAGTACGGCGGGTTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。
SEQ ID No.3：
GACATCAAGATGACCCAGTTTCCATCTTCCATGTATGCATCTTTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGGTATTTAATCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGTAGAGAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGAATATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTACTGTCTACAGTATGATGAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。
and translating the obtained base sequence into an amino acid sequence, and analyzing to obtain the 521I4D5 anti-MLH 1 recombinant rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the anti-MLH 1 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-MLH 1 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：
QVQLQQSGAELVRPGTSVKVSCKASGYAFTDYLIEWVKQWPGQGLEWIGVINPGSGNTDYNEKFRGKATLTADKSSSTAYMHLTSLTSDDSAVYFCTRWVKYGGFFDYWGQGTTLTVSS。
SEQ ID No.5:
DIKMTQFPSSMYASLGERVTITCKASQDINRYLIWFQQKPGKSPKTLIYRVERLVDGVPSRFSGSGSGQEYSLTISSLEYEDMGIYYCLQYDEFPFTFGSGTKLEIK。
example 2
The present example is an immunohistochemical detection of anti-MLH 1 recombinant rabbit monoclonal antibody as primary antibody, the method is as follows:
(1) Sample slice preparation: slicing the tonsil, colon cancer and MLH 1-deleted colon cancer tissue after formalin-fixed paraffin embedding in a constant temperature oven at 60 ℃ for 1-2h, and preserving for later use;
(2) Slice dewaxing: the paraffin section is firstly placed in fresh dimethylbenzene for dewaxing, and is soaked for 2 times, and each time is 10 min;
(3) Slice hydration: sequentially soaking in absolute ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5min for hydration, and washing with purified water for 2 times each for 3 min;
(4) Antigen retrieval: repairing for 3min by using a high-temperature thermal repairing method (if an automatic repairing instrument is used, the repairing can be carried out for 20 min at the high temperature of 98 ℃), naturally cooling the slice to room temperature, then looping the tissue to be detected by using an immunohistochemical pen, and flushing with purified water for 2 times for 3min each time;
(5) Inactivation of endogenous peroxidases: dripping a proper amount of endogenous peroxidase blocking agent to completely cover the tissue, incubating for 10min at room temperature, and washing with purified water for 2 times, each for 3min, and washing with Phosphate Buffer Solution (PBST);
(6) Incubation resistance: adding 100 mu L of 1 mu g/mL 521I4D5 anti-MLH 1 recombinant rabbit monoclonal antibody to completely cover the tissues, placing the tissues in a 37 ℃ incubator for incubation for 1h, and flushing with Phosphate Buffer (PBST) for 3 times for 5min each time;
(7) Secondary antibody incubation: performing secondary antibody incubation according to the instruction of a secondary antibody staining system 3, 3-Diaminobenzidine (DAB) staining solution kit, and washing the plate 3 times with Phosphate Buffer (PBST) after incubation for 5min each time and 1 time with purified water;
(8) DAB color development: preparing 3, 3-Diaminobenzidine (DAB) color development liquid according to the instruction of the 3, 3-Diaminobenzidine (DAB) color development liquid kit, dripping a proper amount of the prepared 3, 3-Diaminobenzidine (DAB) color development liquid until the 3, 3-Diaminobenzidine (DAB) color development liquid completely covers tissues, stopping dyeing after no deepening of color, and flushing with purified water for 3 times;
(9) Hematoxylin counterstain: counterstaining the sections according to hematoxylin manufacturer's instructions and advice, and flushing with Phosphate Buffer (PBST) or tap water to return to blue;
(10) And (3) dehydration and transparency: sequentially soaking 70%,85%,95%,100% gradient alcohol and 3min each time; 2 times of xylene is transparent, and each time is 5min;
(11) Sealing piece: the samples were flaked with neutral gum.
As can be seen from the results of FIG. 1, MLH1 protein is specifically cell nucleus-stained in human tonsil, colon cancer and MLH 1-deleted colon cancer tissues, and the MLH1 recombinant rabbit monoclonal antibody of 521I4D5 clone has better staining effect and darker staining color than the commercially available MLH1 antibody. The 521I4D5 cloned MLH1 recombinant rabbit monoclonal antibody has the characteristics of good specificity, strong positive signals and the like, is easier to evaluate in IHC staining, and is more accurate for detecting and distinguishing cancers.
Example 3
This example shows the measurement of the affinity of 521I4D5 anti-MLH 1 recombinant rabbit monoclonal antibody as follows:
(1) The labeled MLH1 polypeptide (SEQ ID No. 1) was removed from 4℃and returned to room temperature. Diluted to a concentration of 1. Mu.g/ml, incubated at 4℃overnight at 100. Mu.L/well on a 96-well ELISA plate, followed by blocking at 4℃overnight with 2% Bovine Serum Albumin (BSA);
(2) Diluting the MLH1 recombinant rabbit monoclonal antibody cloned by 521I4D5 to an initial concentration of 0.5 mug/mL, sequentially carrying out 2-time gradient dilution, and setting 8 concentration gradients for comparison;
(3) Adding diluted anti-MLH 1 recombinant rabbit monoclonal antibody to a 96-hole ELISA plate with polypeptide according to 100 mu L/hole, covering a sealing plate film, and incubating for 1h at a constant temperature of 37 ℃ to balance the reaction;
(4) Taking out the ELISA plate after the reaction is finished, discarding liquid, washing with purified water for 5 times, and drying the water by beating;
(5) Diluting horseradish peroxidase (HRP) marked goat anti-rabbit IgG according to the second antibody application instruction, adding 100 mu L/hole into an ELISA plate, and incubating at 37 ℃ for 1h to balance the reaction;
(6) Taking out the ELISA plate after the reaction is finished, discarding liquid, washing with purified water for 5 times, and drying the water by beating;
(7) Adding 3,3', 5' -tetramethyl benzidine (TMB) color development solution into the mixture according to 100 mu L/hole, and reacting for 6 minutes at room temperature;
(8) After completion of the reaction, 2M H was added at a concentration of 50. Mu.L/well 2 SO 4 Terminating the color development;
(9) OD values were read at 450nm on a microplate reader, data were collated, and the analysis results are shown in fig. 2 below.
The results show that in 8 concentration gradient tests, the 521I4D5 cloned anti-MLH 1 recombinant rabbit monoclonal antibody has strong affinity and high sensitivity to MLH1 protein molecules, can still reach higher OD value under the condition of lower antibody concentration, and can save the experiment and detection cost.
Example 4
In this example, immunoblotting (Western blotting) of the anti-MLH 1 recombinant rabbit monoclonal antibody 521I4D5 as primary antibody was performed as follows:
(1) Selecting polyvinylidene fluoride (PVDF) membrane of human T lymphocyte leukemia cell (Jurkat) 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% Bovine Serum Albumin (BSA), and shaking at room temperature for 2h; the TBST is a common washing buffer solution and is suitable for experiments such as immunoblotting and the like. It contains three basic components: tris buffer, salt (typically sodium chloride), and surfactant Tween-20.
(2) Incubation resistance: diluting 521I4D5 antibody to 0.5 mug/mL concentration, putting the sealed membrane into the corresponding diluted antibody, and incubating at 4 ℃ with shaking overnight;
(3) The membrane was taken out and washed 3 times (2X 5 min+1X 10 min) in TBST;
(4) 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;
(5) The membrane strip was taken out and washed 4 times (3X 5 min+1X 8 min) in TBST;
(6) 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;
(7) 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 the band size of MLH1 protein in human T lymphocyte leukemia cell (Jurkat) lysate is 84 kDa.
As can be seen from the results of FIG. 3, in the lanes, the 521I4D5 anti-MLH 1 recombinant rabbit monoclonal antibody can specifically recognize the MLH1 protein in human T lymphocyte leukemia cells, and the molecular weight is about 84kDa, which indicates that the 521I4D5 cloned MLH1 recombinant rabbit monoclonal antibody of the invention can specifically recognize the MLH1 protein.
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 (9)
1. An anti-MLH 1 recombinant rabbit monoclonal antibody, which is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.
2. A coding gene for the anti-MLH 1 recombinant rabbit monoclonal antibody of claim 1.
3. The coding gene according to claim 2, characterized in that it comprises: a DNA sequence shown as SEQ ID No.2 for encoding the heavy chain variable region of the anti-MLH 1 recombinant rabbit monoclonal antibody, and a DNA sequence shown as SEQ ID No.3 for encoding the light chain variable region of the anti-MLH 1 recombinant rabbit monoclonal antibody.
4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.
5. An expression vector or recombinant plasmid comprising the nucleic acid molecule of claim 4.
6. A preparation method of an anti-MLH 1 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-MLH 1 recombinant rabbit monoclonal antibody.
7. Use of an anti-MLH 1 recombinant rabbit monoclonal antibody according to claim 1, a coding gene according to claim 2 or 3, a nucleic acid molecule according to claim 4, an expression vector according to claim 5 or a recombinant plasmid for the preparation of an MLH1 detection device.
8. An MLH1 detection kit comprising an anti-MLH 1 recombinant rabbit monoclonal antibody according to claim 1 and an immunohistochemical detection reagent.
9. The MLH1 detection kit of claim 8, comprising: the anti-MLH 1 recombinant rabbit monoclonal antibody, horseradish peroxidase labeled secondary antibody, ethylenediamine tetraacetic acid repair liquid, catalase blocking liquid, 3-diaminobenzidine concentrated solution, 3-diaminobenzidine buffer solution, hematoxylin and bluing liquid.
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| CN1314390A (en) * | 2000-03-22 | 2001-09-26 | 上海博德基因开发有限公司 | New polypeptide-human DNA mispairing repair protein 8 and polynucleotide for coding such polypeptide |
| CN110845612A (en) * | 2019-08-09 | 2020-02-28 | 无锡傲锐东源生物科技有限公司 | Anti-mismatch repair protein MLH1 monoclonal antibody and immunodetection application thereof |
| CN114213533A (en) * | 2021-12-31 | 2022-03-22 | 河南赛诺特生物技术有限公司 | Monoclonal antibody aiming at human mismatch repair protein PMS2, preparation method thereof, immunoassay reagent and application |
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| CN1314390A (en) * | 2000-03-22 | 2001-09-26 | 上海博德基因开发有限公司 | New polypeptide-human DNA mispairing repair protein 8 and polynucleotide for coding such polypeptide |
| CN110845612A (en) * | 2019-08-09 | 2020-02-28 | 无锡傲锐东源生物科技有限公司 | Anti-mismatch repair protein MLH1 monoclonal antibody and immunodetection application thereof |
| CN114213533A (en) * | 2021-12-31 | 2022-03-22 | 河南赛诺特生物技术有限公司 | Monoclonal antibody aiming at human mismatch repair protein PMS2, preparation method thereof, immunoassay reagent and application |
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