CN113930408A - Plano-bamboo-leaf PLA2 protein specific short peptide, anti-Plano-bamboo-leaf PLA2 protein antibody and snake bite detection kit - Google Patents

Plano-bamboo-leaf PLA2 protein specific short peptide, anti-Plano-bamboo-leaf PLA2 protein antibody and snake bite detection kit Download PDF

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CN113930408A
CN113930408A CN202111191571.4A CN202111191571A CN113930408A CN 113930408 A CN113930408 A CN 113930408A CN 202111191571 A CN202111191571 A CN 202111191571A CN 113930408 A CN113930408 A CN 113930408A
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pla
protein
antibody
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monoclonal antibody
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CN113930408B (en
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赖仞
龙承波
吴飞龙
杨敏
吕秋敏
李东升
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Kunming Institute of Zoology of CAS
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Abstract

The invention provides a bamboo leaf green PLA2Protein-specific short peptide and anti-bamboo leaf-green PLA2A protein antibody and snake bite detection kit belongs to the technical field of antibody and immunodetection. Plantain PLA2The amino acid sequence of the protein specificity short peptide is shown as SEQ ID NO. 1. Anti-bamboo leaf green PLA obtained by screening short peptide2Protein monoclonal antibody PLA2-mc8 and Zhuyeqing natural PLA2The protein has higher specific affinity capability. Made of PLA2Snake bite made of protein monoclonal antibodyThe diagnostic kit can be used for diagnosing whether the snake bite is bitten by the ilex latifolia, and greatly improves the diagnosis efficiency and result accuracy of the snake bite by taking antibody-antigen immunoreaction as the detection and diagnosis basis.

Description

Plano-bamboo-leaf PLA2 protein specific short peptide, anti-Plano-bamboo-leaf PLA2 protein antibody and snake bite detection kit
Technical Field
The invention belongs to the technical field of antibody and immunodetection, and particularly relates to a phyllostachys edulis PLA2Protein-specific short peptide and anti-bamboo leaf-green PLA2Protein antibody and snake bite detection kit.
Background
The green bamboo snake is one of the most deadly organisms in the world 10, and the produced toxin is blood circulation toxin. The circulating blood toxicity has many kinds and complex components, mainly including cardiovascular and blood systems, and produces various toxic effects. The toxin expelling amount is small when the green bamboo leaves bite a person, the toxicity is changed into the main effect of hemorrhagic, 15 mg of toxin can be eliminated once, but the average 100 mg can kill the person, so that the person rarely dies, but the person can leave a wound if the person is not treated in time.
At present, the method for identifying the bite of the viper is mainly identified clinically by the main methods of family members or patients such as the identification of the viper atlas, clinical manifestations, snake tooth marks and the like, and the diagnosis error of the methods is very large. In a large number of cases, the snake species are not identified for bite, thereby delaying the valuable treatment window and leading to death. The diagnostic efficiency of snake bite can be greatly improved by taking antibody-antigen immunoreaction as the basis of detection and diagnosis. At present, the anti-snake venom serum varieties in China are few, and the anti-snake venom serum varieties cannot cover all the virulent snake varieties. The toxic components contained in snake venom are very complex and mainly comprise enzyme, neurotoxic polypeptide, bioactive factor and membrane active polypeptide. The snake venom contains at least 10-15 enzymes, which are closely related to bleeding, edema, muscle necrosis and blood coagulation disorder caused by snake injury. Phospholipase A2(PLA2) It is abundant in snake venom. Develop to aim at PLA2The antibody has important significance for rapidly detecting the bite of the green bamboo snake. The lack of effective specific Plantago Cannabina PLA at present2The monoclonal antibody of (1) and the use thereof for screening Phyllostachys nigra PLA2The PLA2 protein-specific short peptide of the monoclonal antibody of (1).
Disclosure of Invention
In view of the above, the present invention aims to provide a bamboo leaf PLA2Protein-specific short peptides capable of being used for specific screening of PLA2A protein monoclonal antibody.
The invention also aims to provide PLA for resisting bamboo leaf green2Protein antibody and snake bite detection kit, realize the snake bite of green bamboo snake and diagnose fast.
The invention provides a bamboo leaf green PLA2The amino acid sequence of the protein specificity short peptide is shown as SEQ ID NO. 1.
The invention provides an anti-bamboo leaf-green PLA obtained by screening the short peptide2Protein monoclonal antibody PLA2-mc8, the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO:2, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO:3, respectively.
The invention provides an ELISA detection kit for treating snake bite of Trimeresurus stejnegeri, which comprises a coating anti-Trimeresurus stegineri PLA2Solid phase carrier of protein polyclonal antibody, enzyme-labeled anti-bamboo leaf-green PLA2Protein monoclonal antibody PLA2-mc8。
Preferably, the anti-bamboo leaf-green PLA2The protein polyclonal antibody is PLA separated and purified from Trimeresurus trifoliatus venom2The protein immune animal is prepared.
Preferably, the phyllostachys nigra PLA2The amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively.
Preferably, the anti-bamboo leaf-green PLA in the solid phase carrier2The coating concentration of the polyclonal antibody of the protein is 1-100 mug/ml.
Preferably, the detection kit further comprises a washing solution, a sample diluent, a substrate, a developing solution and a positive standard substance.
Preferably, the positive standard substance comprises an isolated and purified PLA protein or the Plantago lanceolata PLA2Protein-specific short peptides.
The bamboo leaf green PLA provided by the invention2The amino acid sequence of the protein specificity short peptide is shown as SEQ ID NO. 1. PLA (polylactic acid)2The protein exists in various snake venom, and different sources of snake venom PLA are found through comparison2The protein exists in a conserved region and a hypervariable region, and the PLA provided by the invention2The protein short peptide exists in the specificity of the trimeresurus veneriformis venom, and the short peptide is adopted to screen the trimeresurus veneriformis PLA2The protein monoclonal antibody can realize the specific detection of the bamboo leaf green snake bite.
The invention provides the anti-bamboo leaf-green PLA obtained by screening the short peptide2Protein monoclonal antibody PLA2-mc8, the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO:2, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO:3, respectively. The monoclonal antibody PLA2The-mc 8 has strong specificity, the antiserum secreted by the prepared hybridoma cell strain is subjected to immune combination reaction with five snake venoms (bamboo leaf green, viper, cobra and iron head), and only the bamboo leaf green snake venoms can be specifically combined with the antiserum. Subsequently, antiserum secreted by different hybridoma cell strains and bamboo leaf green PLA2The protein-specific short peptide is subjected to immunological binding reaction, PLA2-mc8、PLA2-mc11 and PLA2-mc14 capable of reacting with PLA2Short peptide antigen binding, and PLA2mC8 has the strongest affinity with PLA2 short peptide antigen, therefore PLA2-mc8 specific binding to Phyllostachys nigra PLA2And the protein ensures the result accuracy of the detection kit.
Drawings
FIG. 1 is a PLA of Trifolium Bambusae2Protein (phospholipaseA)2Access: Q6H3D3.1) NCBI blast results, boxed with PLA2-pep-3 as the specific sequence SEQ ID NO 1;
FIG. 2 is a PLA of Trimeresurus albolabris2The result of protein homology modeling is marked as a PLA2 protein hypervariable region (PLA)2-pep-1、PLA2Pep-2 and PLA2-pep-3)；
FIG. 3 is a PLA of Trifolium Bambusae2Separating and purifying (A) in a protein medium-pressure liquid phase (cation column) and WB results (B) of each peak after purification, wherein the No. 10 peak is PLA2The peak where the protein is located;
FIG. 4 shows crude toxin of Zhuyeqing and PLA of the purified Zhuyeqing2Protein denaturation SDS-PAGE electrophoresis result;
FIG. 5 shows five natural snake venom (A) and natural PLA2Protein (B) screening monoclonal antibody results;
FIG. 6 shows PLA used2-pep-3 polypeptide performing a second round of screening on the screened monoclonal antibody;
FIG. 7 is anti-natural PLA2The detection results of the specificity and space accessibility of the monoclonal antibody and the polyclonal antibody of the protein antigen; wherein A is anti-PLA respectively2Monoclonal antibody PLA2-ELISA and WB detection results for mc 8; b is anti-PLA respectively2ELISA and WB detection results of the polyclonal antibody;
FIG. 8 shows the results of five snake venom detection (ELISA) after successful monoclonal antibody and polyclonal antibody pairing;
FIG. 9 is a standard curve of the concentration measurement of Trimeresurus trifoliatus venom, showing good linearity in the range of 6.25ng/ml to 100 ng/ml.
Detailed Description
The invention provides a bamboo leaf green PLA2The amino acid sequence of the protein-specific short peptide is shown as SEQ ID NO. 1 (INLKLFCKKTSEQC).
In the present invention, PLA of various poisonous snakes2Protein sequences are compared to obtain a section of highly variable amino acid sequence which is conservative in the species of the green bamboo snake and is formed with other poisonous snakes, and the adoption of the amino acid sequence can specifically distinguish the PLA of the green bamboo snake2Proteins and other Snake venom PLA2Protein, for subsequent screening of anti-azulene PLA2Protein antibodies provide a tool.
The invention provides an anti-bamboo leaf-green PLA obtained by screening the short peptide2Protein monoclonal antibody PLA2-mc8, the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO: 2(QVQLRQSGAELVRPGSSVKISCKASGYAFSGYWMNWVKQRPGQGLEWIGQIYPGDGDTIYNGKFKGKVTLTADKSSSTAYIQLSSLTSEDSAVYFCARLTGAFAMDFWGQGTSVTVSSQVQLRQSGAELVRPGSSVKISCKASGYAFSGYWMNWVKQRPGQGLEWIGQIYPGDGDTIYNGKFKGKVTLTADKSSSTAYIQLSSLTSEDSAVYFCARLTGAFAMDFWGQGTSVTVSS), the monoclonal antibody having the amino acid sequence of the light chain variable regionThe sequence is shown as SEQ ID NO:3 (DIQMTQSPAFLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGNSWTWTFGGGTKLEIKDIQMTQSPAFLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGNSWTFGGGTKLEIK).
In the invention, the anti-bamboo leaf-green PLA2Protein monoclonal antibody PLA2-mc8 is secreted by hybridoma cell lines. Five natural snake venom antigens and natural bamboo leaf green PLA2Antigen is respectively coated on solid phase carriers, antiserum secreted by a plurality of screened hybridoma cell strains is subjected to indirect ELISA detection, and the result shows that only the bamboo snake venom and the natural bamboo PLA are present2The antigen is capable of specific binding. And adding bamboo leaf green PLA2The protein specificity short peptide is used as coating antigen to carry out indirect ELISA screening, PLA2-mc8、PLA2-mc11 and PLA2-mc14 is capable of reacting with said PLA2Short peptide antigen binding, wherein PLA2Mc8 has the strongest binding property.
The invention provides an ELISA detection kit for treating snake bite of Trimeresurus stejnegeri, which comprises a coating anti-Trimeresurus stegineri PLA2Solid phase carrier of protein polyclonal antibody, enzyme-labeled anti-bamboo leaf-green PLA2Protein monoclonal antibody PLA2-mc8。
In the invention, the anti-bamboo leaf-green PLA2The protein polyclonal antibody is preferably PLA separated and purified from Trimeresurus trifoliatus venom2The protein immune animal is prepared. The bamboo leaf green PLA2The amino acid sequence of the protein is preferably as shown in SEQ ID NO: 4 (SVIELGKMIFQETGKNPATSYGLYGCNCGPGGRRKPKDATDRCCYVHKCCYKKLTDCDPIKDRYSYSWVNKAIVCGEDNPCPKEMCECDKAVAICFRENLDTYDKKKKINLKLFCKKTSEQC). The number of immunizations of an animal preferably comprises 3, with an interval of 2 weeks. In the immunization, preferably, the isolated and purified PLA is used2The protein and adjuvant are mixed in equal volume and injected into animals. After immunization, antiserum was collected and the antibody titer was determined by indirect ELISA.
In the invention, the anti-bamboo leaf-green PLA in the solid phase carrier2The coating concentration of the polyclonal antibody of the protein is preferably 1-100 mu g/ml, and more preferably5 to 50. mu.g/ml, most preferably 10. mu.g/ml. The coating solvent is preferably 20mM phosphate buffer. The coating method of the present invention is not particularly limited, and a coating method known in the art may be used. After coating, it is preferably closed. The blocking solution is BSA solution with the concentration of 1-10%. The solid phase carrier preferably comprises an enzyme label plate, magnetic particles or a plastic tube and the like.
In the present invention, the enzyme-labeled anti-Trimeresurus PLA2Protein monoclonal antibody PLA2The enzyme in-mc 8 is preferably horseradish peroxidase (HRP). The enzyme-labeled anti-bamboo leaf-green PLA is prepared by2Protein monoclonal antibody PLA2The method for preparing-mc 8 is not particularly limited, and a method for labeling an antibody with an enzyme known in the art may be used.
In the present invention, the detection kit preferably further comprises a washing solution, a sample diluent, a substrate, a developing solution and a positive standard. The positive standard substance preferably comprises separated and purified PLA protein or the bamboo leaf green PLA2Protein-specific short peptides. The washing solution, the sample diluent, the substrate, and the developing solution are not particularly limited in kind, and an ELISA reagent known in the art may be used.
The method for using the kit is not particularly limited, and a double-antibody sandwich ELISA detection method well known in the art can be adopted.
The detection kit provided by the invention is used for detecting the bamboo leaf green and/or the flathead snake venom based on the double-antibody sandwich principle, and the anti-PLA provided by the invention is utilized2Monoclonal antibody and polyclonal antibody of protein have high affinity and strong specificity and are combined with bamboo leaf green PLA2The protein combination ability realizes the rapid diagnosis of the bite of the bamboo leaf green or the soldering iron head, and greatly improves the snake bite detection efficiency and the accuracy of the detection result.
The invention provides a bamboo leaf PLA by combining the following embodiments2Protein-specific short peptide and anti-bamboo leaf-green PLA2Protein antibodies and snake bite detection kits are described in detail, but they should not be construed as limiting the scope of the invention.
The term "anti-bamboo leaf-green PLA2Protein antibody and Zhuyeqing PLA2Antibody and antibody are used interchangeably and refer to the ability to interact with phylline PLA2An antibody to which the protein specifically binds.
The term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
The term "isolated" refers to a substance that is separated from its original environment, which is the natural environment if the substance is natural. If the polynucleotide or protein in a natural state in a living cell is not isolated or purified, the polynucleotide or protein is separated from other substances present in the natural state, and then the polynucleotide or protein is isolated or purified.
The term "hybridoma cell" refers to a cell obtained by fusing myeloma cells and B lymphocytes in the course of producing a monoclonal antibody.
The term "ELISA" refers to a qualitative and quantitative detection method in which a soluble antigen or antibody is bound to a solid phase carrier such as polystyrene and an immunoreaction is carried out by utilizing the binding specificity of the antigen and the antibody.
The invention provides a bamboo leaf-resistant PLA2Protein antibodies capable of reacting with Phyllostachys nigra PLA2The protein is specifically combined, and the antibody is prepared from the natural purified bamboo leaf green PLA2The protein is prepared.
Example 1
Downloading PLA from different snake species from NCBI website2Protein sequences are compared by using comparison software (DNAMAN) to obtain comparison results (shown in figure 1), and the comparison of the Zhuangqing PLA is carried out2The amino acid sequence of the mature protein was found to be divided into conserved regions (white, light grey background) and hypervariable regions (grey, dark grey background). Selection of PLA-localized sites by combining results of homology modeling analysis2Protein surface polypeptide to obtain Phyllostachys nigra PLA2Protein candidate Polypeptide (PLA)2-peptide-1、PLA2-peptide-2 and PLA2-peptide-3), wherein PLA is2-peptide-3 amino acid sequence is shown in SEQ ID NO 1 (INLKLFCKKTSEQC).
Example 2
Bamboo leaf green PLA2Protein specific polypeptide antigen synthesis
Obtaining Phyllostachys nigra PLA by NCBI blast analysis and homologous modeling analysis2Protein specific polypeptide sequence PLA2-peptide-3 (see FIGS. 1 and 2). PLA (polylactic acid)2-peptide-3 antigen polypeptide was synthesized by solid phase synthesis by Gill Biochemical (Shanghai) Co., Ltd. Each short peptide antigen synthesized polypeptide antigen 10mg and purity over 95%.
Example 3
anti-PLA2Specific Polypeptide (PLA)2-peptide-3) polyclonal antibody preparation method, comprising the following steps:
(1) preparation of KLH-polypeptide composite antigen
Synthesizing polypeptide by biochemical synthesis method, and combining with hemocyanin (KLH) to prepare KLH-polypeptide complex antigen. The specific method was carried out according to the kit (K2039-5, BioVision, USA) instructions.
(2) Immunizing animals
The natural solder tip KLH-polypeptide complex antigen was dissolved in saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1mg of antigen) was emulsified in equal amounts of complete Freund's adjuvant (F-5881, sigma) and injected subcutaneously along multiple sites of the back of male New Zealand white rabbits, two rabbits per antigen. After 2 weeks of intervals, booster immunizations were administered intradermally along multiple sites along the back of male New Zealand rabbits using Freund's incomplete adjuvant (F-5506, sigma) emulsified with an equal volume of antigen (0.5ml, containing 0.5mg of antigen). Two booster immunizations were performed at 2 weeks intervals. Rabbit sera were collected 14 days after the second booster immunization. And (3) taking each antigen as a coating antigen, taking serum before immunization as negative control, and detecting the titer of the antibody by adopting an indirect ELISA method. The method comprises the following specific steps:
1) coating: KLH-polypeptide complex antigen (5. mu.g/ml) was diluted with the coating solution and added to a 96-well plate in an amount of 100. mu.l per well and 100. mu.l of the coating solution to a blank well, and coated overnight at 4 ℃.
2) Washing: the next day, the coating solution in the wells was poured off and washed three times with 250. mu.l of wash solution per well for 3-5 minutes each time, and the wells were patted dry as much as possible.
3) And (3) sealing: the blocking solution was applied to 250. mu.l/well for 1h at 37 ℃. Then washed three times and patted dry.
4) Mu.l of rabbit immune serum after gradient dilution is added into the positive hole, and 100. mu.l of sealing solution is added into the negative hole and the blank hole, and the temperature is 37 ℃ for 1 h. Then washed three times and patted dry.
5) 100 μ l of horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibody was added to the positive, negative and blank wells, and incubated for 1h at 37 ℃ in a dark incubator. Then washed three times and patted dry.
6) Color development: after washing, 100. mu.l of color developing solution is added into each well, and the mixture is incubated for 15 to 30 minutes at 37 ℃ in the dark.
7) And (4) terminating: 100. mu.l of stop solution per well was subjected to 450/630nm dual-wavelength reading using a microplate reader.
Wherein, the formula of the solution is as follows:
1. coating liquid: 0.05M carbonate buffer pH 9.6; sodium carbonate 1.59g + sodium bicarbonate 2.93g, dissolved in 1L deionized water.
2. Washing liquid: cells were treated with Phosphate Buffered Saline (PBS) + 0.1% Tween-20. Note: cells were plated with PBS: 0.2g of potassium chloride; potassium dihydrogen phosphate, 0.2 g; sodium chloride, 8 g; 12 g of sodium dihydrogen phosphate monohydrate, 2.16g of deionized water and 1L of deionized water.
3. Blocking solution (antibody dilution): washing solution 1g per 100ml + Bovine Serum Albumin (BSA).
4. Color development liquid: 3,3',5,5' -Tetramethylbenzidine (TMB) single-component color developing solution (Solarbio, Beijing).
5. Stopping liquid: 2mol/L sulfuric acid, 178.3ml water +21.7ml concentrated sulfuric acid, slowly stirring and mixing.
Example 4
The separation and purification method of the bamboo leaf green PLA2 protein comprises the following steps:
a Resource S column was mounted on an FPLC (fast protein liquid chromatography, AKTA pure) instrument, and after the column bed was washed according to the instructions, 50mg of crude bamboo leaf green toxin was dissolved in 2ml of A solution and applied to the well-balanced Resource S column through a sample application valve. Detecting with ultraviolet detector at 280nm and 215nm, collecting each peak, respectively collecting each elution peak, lyophilizing, and storing at-20 deg.C. The method comprises the following specific steps: equilibrating with solution A (flow rate 3ml/min), loading, washing away unbound protein with solution A, performing linear elution with solution B, and collecting each peak. Wherein, the formula of the solution is as follows:
solution A (KCl: 0.2g, KH)2PO4：0.2g，Na2HPO4·12H2O3.47 g, pH 7.2); liquid B (KCl: 0.2g, KH)2PO4：0.2g，Na2HPO4·12H2O:3.47g, NaCl: 58g, pH 7.2) was filtered with a 0.45 μm filter (Millipore) and degassed by ultrasound.
With anti-PLA2Peptide-3 polypeptide antigen antibody was subjected to WB detection to confirm the peak of the target protein (see FIG. 3). Finally, protein purification is detected by performing denaturing SDS-PAGE electrophoresis on the purified Plano-Dianthus PLA2 protein, and the results show that the natural Plano-Dianthus PLA2 protein is obtained by respectively separating and purifying snake venom (see figure 4).
Example 5
Bamboo leaf green PLA2Preparation method of protein monoclonal antibody
1) PLA made of natural bamboo leaf green2And (3) immunizing a mouse by the protein antigen to obtain spleen cells of the immunized mouse.
Natural Trimeresulfonic acid prepared in example 3 was used2The protein was used to immunize 5 Balb/c mice. Wherein Balb/c mice are white-variant laboratory mice, each mouse averagely completes at least 4 times of immunization and 3 times of blood sampling detection, and after the first, second and third blood sampling detection, the target mice with higher titer (screened by the natural antigen) are screened, in the embodiment, 2 mice are screened from 5 mice, and spleen cells of the 2 mice are obtained.
2) Fusing splenocytes and mouse myeloma cells, and screening out PLA which can be mixed with natural bamboo leaf green2Protein antigen-binding hybridoma cell strain.
SP2/0 mouse myeloma cells derived from Balb/c mice were subjected to cell division with spleen cells of 2 selected miceFusing, culturing, observing, detecting and performing negative and positive control tests after fusing to obtain PLA capable of generating anti-bamboo leaf green2Hybridoma cell strain of protein antibody, and further culturing and selecting.
3) Inoculating hybridoma cells into abdominal cavity of mouse for induced culture, culturing to obtain cell culture solution, purifying from the cell culture solution to obtain PLA capable of reacting with folium Bambusae2Anti-bamboo leaf-green PLA with immunoreaction of protein antigen2Protein monoclonal antibody.
Before inoculating hybridoma cells, the mice are injected with 0.5mL of liquid paraffin into the abdominal cavity, and the hybridoma cell suspension prepared by the method is injected into the abdominal cavity of the mice for 2 multiplied by 10 after one week6Per mL, 0.5mL hybridoma cell suspension per mouse was injected.
Collecting ascites of mouse with obviously swollen abdomen under aseptic condition about 7-10 days after inoculating hybridoma cells, centrifuging at 12000g for 10min, removing impurities such as lipid and cells, collecting supernatant obtained by centrifuging, and inactivating at 56 deg.C for 30min to obtain PLA2A protein monoclonal antibody.
Detection of Phyllostachys nigra PLA by indirect ELISA (Enzyme Linked immunosorbent assay)2Protein antibody titers (coating antigen is the native antigen). Specifically, five kinds of natural snake venom and natural PLA are used2Protein as coating antigen screening monoclonal antibody, wherein five natural snake venom antigens are shown as A in figure 5, each well is 100 μ l, the snake venom antigen with concentration of 10 μ g/ml is coated on a 96-well plate, BSA is blocked and washed, the monoclonal antibody is diluted by the antibody secreted by the prepared hybridoma cell strain for incubation, after washing, anti-mouse HRP labeled secondary antibody is added for incubation, after washing, display and OD are performed450And (6) reading.
The specific results are shown in FIG. 5. Only the trimeresurus veneriformis venom can generate specific affinity reaction with the monoclonal antibody, and the monoclonal antibody secreted by different hybridoma cell strains can be combined with natural PLA2The protein undergoes an affinity reaction.
With PLA2The polypeptide-3 is used as a coating antigen to screen monoclonal cell strains and antibodies with high antibody titer. With PLA2-peptide-3 second round screening for polyclonal antibodies for PLA2-Coating peptide-3 antigen (100 μ l per well, 10 μ g/ml) on 96-well plate, blocking with BSA and washing, adding diluted monoclonal antibody for incubation, adding anti-mouse HRP-labeled secondary antibody for incubation after washing, and displaying and OD after washing450And (6) reading.
The results show that PLA2-mc8、PLA2-mc11 and PLA2-mc14 capable of reacting with PLA2-peptide-3 polypeptide antigen binding (FIG. 6). Monoclonal antibody PLA2The-mc 8 mandate the determination of the light chain variable region and the heavy chain variable region by Kinzhi Biotechnology, Inc., Suzhou, with the specific sequences shown in SEQ ID NO 2 and SEQ ID NO 3. Monoclonal antibody PLA2The results of the variable region analysis of-mc 8 are shown in Table 1.
TABLE 1 monoclonal antibody PLA2Sequences of the regions of the variable region of mc8
Figure BDA0003301311960000101
Therefore, in the embodiment, the monoclonal antibody is prepared by an in vivo induction method of an animal inoculated in an abdominal cavity, after the hybridoma cells are cloned successfully, the hybridoma is inoculated in the abdominal cavity of a mouse to induce and generate a large amount of ascites, and simultaneously, a large amount of antibody is secreted in the ascites, so that the concentration of the antibody is high, and the Zhuyeqing PLA with high titer can be conveniently screened2A protein antibody.
Example 6
Anti-natural bamboo leaf green PLA2The preparation method of the protein polyclonal antibody comprises the following specific steps:
mixing natural herba Lophatheri PLA2The protein was dissolved in physiological saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1mg of antigen) was emulsified in equal amounts of complete Freund's adjuvant (F-5881, sigma) and injected subcutaneously along multiple sites of the back of male New Zealand white rabbits, two rabbits per antigen. After 2 weeks of intervals, booster immunizations were administered intradermally along multiple sites along the back of male New Zealand rabbits using Freund's incomplete adjuvant (F-5506, sigma) emulsified with an equal volume of antigen (0.5ml, containing 0.5mg of antigen). The time interval is 2 weeks for two timesThe secondary enhancement of immunity. Rabbit sera were collected 14 days after the second booster immunization.
Example 7
Anti-bamboo leaf-green PLA2Protein monoclonal antibody and anti-bamboo leaf-green PLA2The protein polyclonal antibody is respectively combined with the bamboo leaf PLA2 protein antigen and five kinds of snake venom (bamboo leaf, adder, viper, cobra and iron head) to detect, and the specific steps are the same as the steps of 'indirect ELISA' method for detecting the antibody titer in the embodiment 5. Meanwhile, the specificity and space accessibility of the antibody are detected by a western blotting method (WB).
The results are shown in FIG. 7. Wherein A and B are each anti-PLA2Monoclonal antibody PLA2-mc8 and anti-PLA2ELISA and WB detection of polyclonal antigens. ELISA and WB results show that anti-PLA2Monoclonal antibody PLA2-mc8 and anti-PLA2Polyclonal antibodies all have good specificity.
Example 8
Detection of monoclonal antibody and polyclonal antibody pairing
The specific method of antibody pairing is as follows. Coating: coating solution the anti-natural PLA2 polyclonal antibody is diluted to 10 mu g/ml, each hole is 100 mu l, and the coating is carried out overnight at 4 ℃; and (3) sealing: discarding the coating solution, washing with PBST for 3 times, 5min each time, and incubating at 37 deg.C for 1h with 200 μ l of blocking solution per well; incubation of the samples: discarding the blocking solution, washing with PBST for 3 times, each time for 5min, diluting five kinds of crude toxins (bamboo leaf green, viper, cobra and iron head) with PBS (PBS) at the concentrations of 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml and 7.8125ng/ml, and adding 100 μ l per hole. Incubating at 37 ℃ for 1 h; incubation of PLA2 monoclonal antibody: samples were discarded, PBST washed 3 times for 5min each, and HRP-bearing monoclonal antibody was incubated at a dilution ratio of 1: 3000, adding 100 mul into each hole, and incubating for 1h at 37 ℃; and (3) developing: discarding the monoclonal antibody, washing with PBST for 3 times, adding 100 μ l TMB color development solution, and incubating at 37 deg.C in dark for 10 min; and (4) terminating: mu.l of stop buffer was added to each well and the absorbance at 450nm was measured for each well.
The results are shown in FIG. 8. The result shows that the pairing mode with the polyclonal antibody as the coating antibody and the monoclonal antibody as the detection antibody is the optimal pairing mode.
Example 9
Method for establishing standard curve of trimeresurus veneriformis venom measurement by double-antibody sandwich ELISA method
Anti-snake venom PLA after PBS dilution2Protein polyclonal antibody (10. mu.g/ml) was added to a 96-well plate in an amount of 100. mu.l per well and 100. mu.l of coating solution to a blank well, and coated at 37 ℃ for 1 hour; pouring out the coating liquid in the plate holes, washing three times by using washing liquid, wherein each time is 250 mu l, and each time is 5 minutes, and drying the plate holes as much as possible; sealing the solution in each hole by 250 mu l for 1 hour at 37 ℃, then washing for three times, and patting to dry; adding 100 μ l diluted snake venom into the positive well, adding 100 μ l blocking solution into the negative well and the blank well, incubating at 37 deg.C for 1 hr, washing for three times, and patting to dry; add 100. mu.l Horse Radish Peroxidase (HRP) labeled anti-snake venom PLA to the positive, negative and blank wells2Protein (the concentration of the antibody stock solution is 1mg/ml, diluted according to a ratio of 1: 3000), incubating for 1h in a dark incubator at 37 ℃, then washing for three times and patting dry; adding a developing solution into each 100 mu l of washed wells, and incubating for 25 minutes at 37 ℃ in a dark place; 100. mu.l of stop solution per well was subjected to 450/630nm dual-wavelength reading using a microplate reader.
The standard curve of the trimeresurus snake venom is shown in FIG. 9. The regression equation is that y is 0.01131x +0.1784, wherein R2＝0.996。
Example 10
A kit for detecting trimeresurus venulosus by double-antibody sandwich ELISA comprises the following components:
coated with anti-bamboo leaf-green PLA2A solid phase support for a protein polyclonal antibody; the coating concentration is 10 mug/ml;
horse radish peroxidase labeled anti-bamboo leaf-green PLA2A protein monoclonal antibody;
diluent, chromogenic substrate, stop solution, sealing film and positive standard substance, wherein the positive standard substance is natural PLA of bamboo leaf green2The protein solution is prepared by the following steps:
1. preparing a crude toxin solution, specifically, respectively taking out a proper amount of laboratory-stored phyllostachys edulis freeze-dried powder, and dissolving with a proper volume of ultrapure water to obtain the crude toxin solution.
2. The crude venom solution samples were diluted 5-fold with phosphate buffered saline as described above (solution A). Then filtering with a 0.22 mu m filter membrane, loading the filtrate on a Resource S column, and eluting with a linear gradient of 0-1 mol/L NaCl solution. Then, the elution peaks were detected and collected at wavelengths of 280nm and 215nm, and lyophilized until a native PLA2 protein standard with a purity of greater than 95% was obtained.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Kunming animal research institute of Chinese academy of sciences
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Claims (8)

1. Plantain PLA2The protein specificity short peptide is characterized in that the amino acid sequence of the short peptide is shown as SEQ ID NO. 1.
2. A anti-Trimeresurus PLA screened from the oligopeptide of claim 12Protein monoclonal antibody PLA2-mc8, wherein the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO:2, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO:3, respectively.
3. An ELISA detection kit for treating snake bite is characterized by comprising a coating anti-bamboo leaf green PLA2Solid support for polyclonal antibody against protein, enzyme-labeled anti-Trimeresurus PLA according to claim 22Protein monoclonal antibody PLA2-mc8。
4. The detection kit according to claim 3, wherein the anti-Trimeresurus pustus PLA is2The protein polyclonal antibody is PLA separated and purified from Trimeresurus trifoliatus venom2The protein immune animal is prepared.
5. The detection kit according to claim 4, wherein the Phyllostachys nigra PLA is2The amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively.
6. The detection kit as claimed in claim 3, wherein the solid phase carrier is anti-Phyllostachys nigra PLA2The coating concentration of the polyclonal antibody of the protein is 1-100 mug/ml.
7. The detection kit according to any one of claims 3 to 6, characterized in that the detection kit further comprises a washing solution, a sample diluent, a substrate, a developing solution and a positive standard.
8. The test kit of claim 7, wherein the positive standard comprises isolated and purified PLA2Protein or a Phyllostachys nigra PLA as claimed in claim 12Protein-specific short peptides.
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CN107987164A (en) * 2017-12-18 2018-05-04 青岛珅奥基生物工程有限公司 36 antigen polypeptides of estrogen receptor ER- α and its monoclonal antibody
CN110317270A (en) * 2019-05-09 2019-10-11 中国科学院昆明动物研究所 Antitoxin snake PLA2Protein antibodies and its application
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