CN116333112B - anti-SMA recombinant rabbit monoclonal antibody and application thereof - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to an anti-SMA recombinant rabbit monoclonal antibody and application thereof, wherein the anti-SMA recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and 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. Compared with the commercially available anti-SMA monoclonal antibody, the anti-SMA recombinant rabbit monoclonal antibody provided by the invention has higher affinity with SMA protein, can identify and detect the expression of the SMA protein on tumor cells or immune cells with high specificity and high sensitivity, 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, 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-SMA recombinant rabbit monoclonal antibody and application thereof, in particular to application in immunohistochemical detection.
Background
alpha-SMA (alpha-smooth muscle actin, or ACTA 2) is a subtype of actin with a molecular weight of 42 kDa. alpha-SMA is only transiently expressed in cardiac and skeletal muscle cells during embryonic development and is highly specific for adult smooth muscle cells, so the use of SMA antibodies helps to identify smooth muscle cells and myofibroblasts in normal or cancerous tissue, while being valuable for the identification of smooth muscle tumors, leiomyosarcoma, and multiple adenocarcinomas. Myoepithelial cells can be identified in normal or diseased breast, salivary and sweat glands, perhaps helping to eliminate the possibility of tumor invasion. Therefore, the screening of a high-sensitivity and high-specificity anti-SMA antibody plays a very important role in identifying and detecting the expression of SMA protein on tumor cells or immune 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-SMA recombinant rabbit monoclonal antibody and application thereof, wherein the anti-SMA recombinant rabbit monoclonal antibody has wide application, can accurately identify SMA expression, can well detect SMA protein expression on tumor cells or immune cells through immunohistochemical detection of various different tissues, and can be applied to detection and screening fields such as Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-SMA recombinant rabbit monoclonal antibody, a preparation method, application of the anti-SMA recombinant rabbit monoclonal antibody in an SMA 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 invention provides an anti-SMA recombinant rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being 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-SMA recombinant rabbit monoclonal antibody (SMA rabbit source antibody) can be used for immunohistochemical detection, and can identify and detect the expression of SMA protein on tumor cells or immune cells with high specificity and high sensitivity.
The anti-SMA monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-SMA recombinant rabbit monoclonal antibody provided by the invention is produced by eukaryotic expression of 293 cells through rabbit hybridoma fusion screening. When the anti-SMA 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 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 a heavy chain amino acid sequence and a light chain amino acid sequence 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 SMA protein.
The anti-SMA monoclonal antibody can recognize recombinant SMA antigen proteins and SMA molecules on tumor cells and immune cells; the anti-SMA monoclonal antibodies may also be used in immunohistochemical pathological diagnostic agents.
In a second aspect, the invention provides a coding gene for coding the above anti-SMA recombinant rabbit monoclonal antibody.
Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2, and is used for coding a heavy chain variable region of the anti-SMA 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-SMA recombinant rabbit monoclonal antibody.
In a third aspect, the invention relates to a nucleic acid molecule comprising a coding gene for said anti-SMA 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 invention provides a method for preparing an anti-SMA recombinant rabbit monoclonal antibody, comprising the steps of transfecting cells with the expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-SMA recombinant rabbit monoclonal antibody.
More preferably, the preparation method comprises the steps of:
(1) Immunization of rabbits: firstly analyzing the SMA 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 SMA on a cell membrane, 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: obtaining nucleotide sequences of an antibody heavy chain variable region and an antibody light chain variable region by using a specific primer 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-SMA recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid is applied to the preparation of SMA protein molecule detection devices. Such detection devices include, but are not limited to, SMA detection kits, antibody chips, or antibody probes, among others.
In a seventh aspect, the invention also provides an SMA detection kit comprising an anti-SMA recombinant rabbit monoclonal antibody as described above and an immunohistochemical detection reagent.
Preferably, the SMA detection kit comprises: the anti-SMA recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, EDTA repair liquid, catalase blocking liquid, DAB concentrated liquid, DAB buffer liquid, hematoxylin and bluing liquid.
In the case of immunohistochemical detection, the detection steps comprise dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, sealing and microscopic examination, and the like.
(III) beneficial effects
The anti-SMA recombinant rabbit monoclonal antibody provided by the invention has high specificity and high sensitivity in combination with SMA protein molecules, can specifically identify and detect the expression of SMA protein on tumor cells or immune cells, and is positive and high in expression when detecting SMA protein, so that 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 favorable for obtaining accurate assessment and detection results. The 103B8G8 cloned SMA recombinant rabbit monoclonal antibody has the characteristics of good specificity, strong positive signals and the like, has more obvious dyeing effect in IHC dyeing, and is favorable for accurately detecting and distinguishing cancers.
Drawings
FIG. 1 is a graph showing the results of immunohistochemical detection of anti-SMA monoclonal antibodies and commercially available antibodies prepared in the present invention in human breast cancer tissue.
FIG. 2 is a statistical plot of potency detection of the 103B8G8 anti-SMA monoclonal antibodies and commercially available antibodies of the present invention at 8 gradient concentrations.
FIG. 3 shows the result of immunoblotting (Western blotting) detection of the anti-SMA recombinant rabbit monoclonal antibody 103B8G8 as the primary antibody to verify the recognition capability of the anti-SMA recombinant rabbit monoclonal antibody on SMA 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-SMA recombinant rabbit monoclonal antibodies, comprising the steps of:
(1) Antigen preparation
The specific sequence of the SMA antigen is shown as SEQ ID NO. 1.
SEQ ID No. 1：YDEAGPSIVHRKCF。
The polypeptide sequence is selected by analyzing the SMA molecular sequence according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure of SMA protein molecules on cell membranes. 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
Complete antigen is prepared by coupling with KLH or OVA, and is used as immunogen to immunize rabbits.
(2) Immunization
Mixing and emulsifying complete antigen containing polypeptide sequence of SEQ ID NO.1 (SMA antigen) with complete Freund's adjuvant (1:1), immunizing multiple New Zealand white rabbits respectively by subcutaneous injection, emulsifying SMA antigen containing the polypeptide of SEQ ID NO.1 with incomplete Freund's adjuvant (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 of the SEQ ID NO.1 antigen for the next cell fusion.
(3) Cell fusion
Preparing sp2/0 myeloma cells of murine origin in advance, and allowing the sp2/0 myeloma cells to fuse
In the logarithmic growth phase. 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 by ELISA test using SMA 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 103B8G8.
(5) Sequencing the antibody secreted by the screened hybridoma cell strain
Total RNA was isolated from 103B8G8 hybridoma cells according to the reagent TriZol instructions, reverse transcribed into cDNA according to TIANScript first strand cDNA synthesis kit instructions, 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 pore plates, 1.5ml of culture medium is added into each pore according to the required quantity, and the density is 3 x 10 6 And each ml.
Mixing the eukaryotic expression vector and PEI according to the proportion 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, ELISA detection is carried out on transfected 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 heavy chain variable region amino acid sequence of the 103B8G8 anti-SMA recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the light chain variable region amino acid sequence of the anti-SMA 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：
tgtcagtcggtggaggagtccggaggaggcctggtaacgcctgggacacccctgacactcacctgcacagtctctggattctccctcagtaacaatgcaatgagctgggtccgccaggctccagggaaggggctggaatggatcggagtcattggttatagtggtaggatatactacgcgagctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaagatcaccagtccgacaaccgaggacacggccacctatttctgtgtcagatggatttatggtgataataacttgtggggccaaggcaccctggtcaccgtctcctcaa。
SEQ ID No.3：
gcccaagtgctgacccagactccatcctccgtgtctgcggctgtgggaggcacagtcaccatcaactgccaggccagtcagagtgtttatgataacaactacttatcctggtatcagcagaaagcagggcagcctcccaagcgcctgatctatggtgcatccactctggcaactggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgacgatgctgccacttactactgtctaggcaattatgattgtagtagtgctgattgtgctgctttcggcggagggaccgaggtggtggtcaaag。
and translating the obtained base sequence into an amino acid sequence, and analyzing to obtain the 103B8G8 anti-SMA recombinant rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the anti-SMA 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-SMA 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：
CQSVEESGGGLVTPGTPLTLTCTVSGFSLSNNAMSWVRQAPGKGLEWIGVIGYSGRIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRWIYGDNNLWGQGTLVTVSS。
SEQ ID No.5:
AQVLTQTPSSVSAAVGGTVTINCQASQSVYDNNYLSWYQQKAGQPPKRLIYGASTLATGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGNYDCSSADCAAFGGGTEVVVK。
example 2
The present example is an immunohistochemical detection of anti-SMA recombinant rabbit monoclonal antibody as primary antibody, the method is as follows:
(1) Sample slice preparation: the human breast cancer slices after being embedded by formalin fixed paraffin are baked for 1-2 hours in a constant temperature oven at 60 ℃ and stored for standby.
(2) Slice dewaxing: paraffin sections were first dewaxed in fresh xylene and soaked 2 times for 10min each.
(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: 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.
(6) Incubation resistance: 100. Mu.L of 10ng/mL 103B8G8 anti-SMA recombinant rabbit monoclonal antibody and commercially available SMA antibody were added to each of the two samples to completely cover the tissue, and the samples were incubated in an incubator at 37℃for 1h with 3 washes of PBST for 5min each.
(7) 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.
(8) DAB color development: 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.
(9) Hematoxylin counterstain: counterstaining the sections according to hematoxylin manufacturer instructions and advice, PBST or tap water washing to return to blue.
(10) And (3) dehydration and transparency: sequentially soaking 70%,85%,95%,100% gradient alcohol and 3min each time; 2 times the xylene is transparent, 5min each time.
(11) Sealing piece: the samples were flaked with neutral gum.
As shown by the results in FIG. 1, SMA protein stained specifically in human breast cancer tissue, and the SMA antibody of clone 103B8G8 stained better (darker color) than the commercially available SMA antibody. Therefore, the 103B8G8 cloned SMA recombinant rabbit monoclonal antibody has the characteristics of good specificity, strong positive signals and the like, is more obvious in IHC staining, and is more accurate in detecting and distinguishing cancers.
Example 3
This example is an assay for affinity for a 103B8G8 anti-SMA recombinant rabbit monoclonal antibody, as follows:
(1) The labeled SMA polypeptide (SEQ ID NO: 1) was removed from 4℃and allowed to return to room temperature. 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.
(2) The SMA recombinant rabbit monoclonal antibody cloned by 103B8G8 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.
(3) The diluted anti-SMA recombinant rabbit monoclonal antibodies are respectively added to a 96-well ELISA plate with polypeptides according to 100 mu L/well, a sealing plate film is covered, and the temperature is kept constant at 37 ℃ for 1 hour, so that the reaction reaches equilibrium.
(4) 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.
(5) 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.
(6) 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.
(7) TMB color-developing solution was added at 100. Mu.L/well, and the reaction was carried out at room temperature for 6 minutes.
(8) After completion of the reaction, 2M H was added at a concentration of 50. Mu.L/well 2 SO 4 The color development was terminated.
(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 anti-SMA recombinant rabbit monoclonal antibody cloned by 103B8G8 has strong affinity and high sensitivity to SMA protein molecules, can reach a higher OD value under the condition of lower antibody concentration (especially 31.25-125 ng/mL), and is beneficial to ensuring the detection accuracy and saving the experiment and detection cost.
Example 4
This example is a immunoblotting (Western blotting) assay of anti-SMA recombinant rabbit monoclonal antibody 103B8G8 as primary antibody, as follows:
(1) Selecting PVDF membrane of corresponding cell (human skeletal muscle) 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 the 103B8G8 antibody to a concentration of 0.5 mug/mL, putting the blocked film into the corresponding diluted antibody, and incubating at 4 ℃ with shaking for 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: equal amounts of Luminol/enhancer solution (Luminol reagent/enhancer solution) and Peroxide solution (peroxide solution) diluted 5-fold with pure water were mixed in the same vessel, membrane strips were added, and incubated 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.
Lane 1 in fig. 3: human skeletal muscle tissue lysate h.skeletal musculature; stripe size: 51 kDa. As can be seen from the figure, in the lane, the 103B8G8 anti-SMA recombinant rabbit monoclonal antibody can specifically recognize the SMA protein which is overexpressed in the human skeletal muscle tissue lysate, and the molecular weight is about 42kDa, which indicates that the 103B8G8 cloned SMA recombinant rabbit monoclonal antibody can specifically recognize the SMA 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-SMA recombinant rabbit monoclonal antibody 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-SMA recombinant rabbit monoclonal antibody of claim 1.
3. The coding gene according to claim 2, characterized in that it comprises: a DNA sequence as set forth in SEQ ID No.2 for encoding the heavy chain variable region of said anti-SMA recombinant rabbit monoclonal antibody; and a DNA sequence shown as SEQ ID No.3 for encoding a light chain variable region of the anti-SMA recombinant rabbit monoclonal antibody.
4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.
5. An expression vector comprising the nucleic acid molecule of claim 4.
6. A preparation method of an anti-SMA 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-SMA recombinant rabbit monoclonal antibody.
7. Use of the anti-SMA recombinant rabbit monoclonal antibody of claim 1 in the preparation of SMA detection kit.
8. An SMA detection kit comprising the anti-SMA recombinant rabbit monoclonal antibody of claim 1 and an immunohistochemical detection reagent.
9. The SMA detection kit of claim 8, comprising: the anti-SMA recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, EDTA repair liquid, catalase blocking liquid, DAB concentrated liquid, DAB buffer liquid, hematoxylin and bluing liquid.
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