CN113930408B - Bamboo leaf green PLA2 protein specific short peptide, anti-bamboo leaf green PLA2 protein antibody and snake bite detection kit - Google Patents

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

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CN113930408B
CN113930408B CN202111191571.4A CN202111191571A CN113930408B CN 113930408 B CN113930408 B CN 113930408B CN 202111191571 A CN202111191571 A CN 202111191571A CN 113930408 B CN113930408 B CN 113930408B
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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 green bamboo PLA 2 Protein-specific short peptide and anti-Trimeresurus albolabris PLA 2 Protein antibody and snake bite detection kit, which belongs to the technical field of antibody and immunodetection. Green bamboo PLA 2 The amino acid sequence of the protein specific short peptide is shown as SEQ ID NO. 1. anti-Trimeresurus albolabris PLA obtained by screening with the short peptide 2 Protein monoclonal antibody PLA 2 -mc8 and Trimeresurus chinensis natural PLA 2 The protein has higher specific affinity. From PLA 2 The snake bite diagnosis kit prepared from the protein monoclonal antibody can be used for diagnosing whether snake bite is bitten by bamboo leaves, and the immune reaction of the antibody antigen is used as a detection diagnosis basis, so that the snake bite diagnosis efficiency and the result accuracy are greatly improved.

Description

Bamboo leaf green PLA2 protein specific short peptide, anti-bamboo leaf green PLA2 protein antibody and snake bite detection kit
Technical Field
The invention belongs to the technical field of antibodies and immunodetection, and in particular relates to a green bamboo PLA 2 Protein-specific short peptide and anti-Trimeresurus albolabris PLA 2 Protein antibodies and snake bite detection kits.
Background
The garter snake is one of the most deadly organisms in the world 10, and the toxin produced is blood circulation toxin. The blood circulation has various toxic materials, complex components, mainly cardiovascular and blood systems, and various toxic effects. The toxin expelling amount is small when the bamboo leaves bitten by the people, the toxin is mainly changed from hemorrhagic, 15 mg of toxin can be expelled once, but the average 100 mg of toxin can be killed, so that the death of the people is little, but if the treatment is not timely carried out, the disability can be left.
The existing method for identifying the venomous snake bite mainly depends on identification of family members or patients on the venomous snake atlas, clinical manifestations, snake tooth marks and other main methods for identification in clinic, and the diagnosis errors of the methods are very large. There are a number of cases where the bite snake species could not be identified, delaying the precious treatment window time and death. The immune reaction of the antibody antigen is taken as a detection and diagnosis basis, so that the snake bite diagnosis efficiency can be greatly improved. At present, the antivenins in China are few in variety, and the variety of the antivenins cannot cover all the species of the severe snakes. The venom contains very complex toxic components, mainly including enzymes, neurotoxic polypeptides, bioactive factors and membrane active polypeptides. The snake venom contains at least 10-15 enzymes, which have close relation with bleeding, oedema, muscle necrosis and blood coagulation disorder caused by snake bite. Phospholipase A 2 (PLA 2 ) The snake venom is rich in snake venom. Is developed to target PLA 2 The antibody of the kit has important significance for rapidly detecting the bites of the green snakes of the bamboo leaves. At present, the effective specificity of the green bamboo PLA is lacking 2 Monoclonal antibodies of (2) and use for screening of Trimeresurus albolabris PLA 2 PLA2 protein-specific short peptides of the monoclonal antibodies of (a).
Disclosure of Invention
Accordingly, the present invention aims to provide a green bamboo PLA 2 Protein-specific short peptides, which can be used for specific screening of PLA 2 Protein monoclonal antibodies.
The invention also aims to provide the anti-bamboo leaf green PLA 2 Protein antibody and snake bite detection kit, realizes the quick diagnosis of the snake bite of the green bamboo leaves.
The invention provides a green bamboo PLA 2 The amino acid sequence of the protein specific short peptide is shown as SEQ ID NO. 1.
The invention provides an anti-Trimeresurus albolabris PLA screened by the short peptide 2 Protein monoclonal antibody PLA 2 -mc8, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is set forth in SEQ ID NO:2, the amino acid sequence of the light chain variable region of the monoclonal antibodyThe sequences are shown in SEQ ID NO:3.
The invention provides an ELISA detection kit for snake bite of green bamboo leaves, which comprises coating anti-green bamboo leaves PLA 2 Solid phase carrier of polyclonal antibody of protein and enzyme labeled anti-Trimeresurus albolabris PLA 2 Protein monoclonal antibody PLA 2 -mc8。
Preferably, the anti-Trimeresurus albolabris PLA 2 The protein polyclonal antibody is PLA separated and purified from Trimeresurus chinensis venom 2 The protein immune animal is prepared.
Preferably, the bamboo leaf green PLA 2 The amino acid sequence of the protein is shown in SEQ ID NO: 4.
Preferably, the solid phase carrier is resistant to the green-bamboo PLA 2 The 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 chromogenic solution and a positive standard substance.
Preferably, the positive standard comprises an isolated and purified PLA protein or the Trimeresurus chinensis PLA 2 Protein-specific short peptides.
The invention provides a green bamboo PLA 2 The amino acid sequence of the protein specific short peptide is shown as SEQ ID NO. 1. PLA (polylactic acid) 2 The protein exists in various snake venom, and different sources of snake venom PLA are found by comparison 2 Protein storage conservation region and hypervariable region, PLA provided by the invention 2 The protein short peptide is specifically existing in the venom of the Trimeresurus albolabris, and the short peptide is adopted to screen Trimeresurus albolabris PLA 2 The protein monoclonal antibody can realize the specific detection of the green bamboo snake bite.
The invention provides an anti-Trimeresurus albolabris PLA screened by the short peptide 2 Protein monoclonal antibody PLA 2 -mc8, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is set forth in SEQ ID NO:2, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO:3. The monoclonal antibody PLA 2 The mc8 has strong specificity, and the antiserum secreted by the hybridoma cell strain is mixed with five kinds of venom (Trimeresurus chinensis, viper, agkistrodon halys, cobra and cautery)Iron head) and only the Trimeresurus albolabris venom can be specifically combined with antiserum. Antisera secreted by different hybridoma cell lines are then used for preparing Trimeresurus albolla 2 Protein specific short peptide for immune binding reaction, PLA 2 -mc8、PLA 2 -mc11 and PLA 2 -mc14 is capable of associating with PLA 2 Short peptide antigen binding, and PLA 2 -mc8 has the highest affinity for PLA2 short peptide antigen, thus PLA 2 -mc8 specifically binds to Trimeresurus albolabris PLA 2 And the protein ensures the accuracy of the result of the detection kit.
Drawings
FIG. 1 shows a green PLA of bamboo leaves 2 Protein (phospholipaseA) 2 Access Q6H3D3.1) NCBI blast results, box marks PLA2-pep-3 as specific sequence SEQ ID NO 1;
FIG. 2 shows a green PLA of bamboo leaves 2 Protein homology modeling results show that the marked region is PLA2 protein hypervariable region (PLA) 2 -pep-1、PLA 2 Pep-2 and PLA 2 -pep-3)；
FIG. 3 shows a green PLA of bamboo leaves 2 Separating and purifying (A) protein medium-pressure liquid phase (cation column) and purifying each peak WB result (B), wherein the 10 # peak is PLA 2 Peak of protein;
FIG. 4 shows crude toxicity of Trimeresurus chinensis and purified Trimeresurus chinensis PLA 2 Protein denaturation SDS-PAGE electrophoresis result;
FIG. 5 shows the use of five natural snake venom (A) and natural PLA 2 Screening monoclonal antibody results with protein (B);
FIG. 6 shows the use of PLA 2 -subjecting the screened monoclonal antibodies to a second round of screening results with pep-3 polypeptides;
FIG. 7 is an anti-natural PLA 2 The specificity and space accessibility detection results of the monoclonal antibodies and the polyclonal antibodies of the protein antigen; wherein A is respectively PLA resistant 2 Monoclonal antibody PLA 2 ELISA and WB detection results for mc 8; b is PLA resistant respectively 2 ELISA and WB detection results of polyclonal antibodies;
FIG. 8 shows the results of five snake venom determinations (ELISA) after successful pairing of monoclonal and polyclonal antibodies;
FIG. 9 shows a standard curve for concentration detection of Trimeresurus chinensis venom, and shows that the curve has good linearity in the range of 6.25ng/ml to 100 ng/ml.
Detailed Description
The invention provides a green bamboo PLA 2 The amino acid sequence of the protein specific short peptide is shown as SEQ ID NO. 1 (INLKLFCKKTSEQC).
In the present invention, PLA from various kinds of venomous snakes 2 The protein sequences are compared to obtain a section of amino acid sequence which is highly mutated with other venomous snakes and is conserved in the species of the Trimeresurus albolabris, and the amino acid sequence can be adopted to specifically distinguish the Trimeresurus albolabris PLA 2 Protein and other snake venom PLAs 2 Protein, for subsequent screening of anti-Trimeresurus albolabris PLA 2 Protein antibodies provide a tool.
The invention provides an anti-Trimeresurus albolabris PLA screened by the short peptide 2 Protein monoclonal antibody PLA 2 -mc8, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is set forth in SEQ ID NO:2 (QVQLRQSGAELVRPGSSVKISCKASGYAFSGYWMNWVKQRPGQGLEWIGQIYPGDGDTIYNGKFKGKVTLTADKSSSTAYIQLSSLTSEDSAVYFCARLTGAFAMDFWGQGTSVTVSSQVQLRQSGAELVRPGSSVKISCKASGYAFSGYWMNWVKQRPGQGLEWIGQIYPGDGDTIYNGKFKGKVTLTADKSSSTAYIQLSSLTSEDSAVYFCARLTGAFAMDFWGQGTSVTVSS), the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:3 (DIQMTQSPAFLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGNSWTWTFGGGTKLEIKDIQMTQSPAFLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGNSWTFGGGTKLEIK).
In the invention, the anti-Trimeresurus albolabris PLA 2 Protein monoclonal antibody PLA 2 -mc8 is secreted by hybridoma cell lines. Five natural snake venom antigens and natural Trimeresurus albolabris PLA 2 The antigen is respectively coated on a solid phase carrier, and the antiserum secreted by the screened various hybridoma cell strains is indirectly detected by ELISA, and the result shows that the antiserum is only prepared from the Trimeresurus venom and the natural Trimeresurus PLA 2 Antigens are capable of specific binding. Then bamboo leaves green PLA 2 Indirect ELISA screening and PLA (polylactic acid) are carried out by taking protein specific short peptide as coating antigen 2 -mc8、PLA 2 -mc11PLA 2 -mc14 is capable of associating with said PLA 2 Short peptide antigen binding, wherein PLA 2 -mc8 has the strongest binding properties.
The invention provides an ELISA detection kit for snake bite of green bamboo leaves, which comprises coating anti-green bamboo leaves PLA 2 Solid phase carrier of polyclonal antibody of protein and enzyme labeled anti-Trimeresurus albolabris PLA 2 Protein monoclonal antibody PLA 2 -mc8。
In the invention, the anti-Trimeresurus albolabris PLA 2 The protein polyclonal antibody is preferably PLA separated and purified from Trimeresurus chinensis venom 2 The protein immune animal is prepared. Green bamboo PLA 2 The amino acid sequence of the protein is preferably as set forth in SEQ ID NO:4 (SVIELGKMIFQETGKNPATSYGLYGCNCGPGGRRKPKDATDRCCYVHKCCYKKLTDCDPIKDRYSYSWVNKAIVCGEDNPCPKEMCECDKAVAICFRENLDTYDKKKKINLKLFCKKTSEQC). The number of times the animal is immunized preferably includes 3 times, with an interval of 2 weeks. In the case of such immunization, it is preferred that the isolated and purified PLA 2 The protein was mixed with an adjuvant in equal volumes and injected into animals. After immunization, antisera were collected and tested for antibody titers by indirect ELISA.
In the invention, the solid phase carrier is resistant to the green-bamboo PLA 2 The coating concentration of the polyclonal antibody of the protein is preferably 1 to 100. Mu.g/ml, more preferably 5 to 50. Mu.g/ml, most preferably 10. Mu.g/ml. The coating solvent is preferably 20mM phosphate buffer. The method of coating is not particularly limited, and coating methods known in the art may be employed. After coating, sealing is preferably carried out. The blocking solution is a BSA solution with a concentration of 1% -10%. The solid phase carrier preferably comprises an ELISA plate, magnetic particles or plastic tubes, etc.
In the present invention, the enzyme-labeled anti-Trimeresurus albolabris PLA 2 Protein monoclonal antibody PLA 2 Preferably, the enzyme in mc8 is horseradish peroxidase (HRP). The enzyme marked anti-bamboo leaf green PLA of the invention 2 Protein monoclonal antibody PLA 2 The method for producing the-mc 8 is not particularly limited, and the method for labeling the antibody with an enzyme known in the art may be used.
In the present invention, the detection kit preferably further comprises a washing solutionSample dilution, substrate, color development solution and positive standard. The positive standard preferably comprises an isolated and purified PLA protein or the Trimeresurus chinensis PLA 2 Protein-specific short peptides. The types of the washing solution, the sample dilution solution, the substrate and the color developing solution are not particularly limited, and ELISA reagents well known in the art can be used.
The method of using the kit is not particularly limited, and the double-antibody sandwich ELISA detection method well known in the art can be adopted.
The detection kit provided by the invention detects the venom of the green bamboo and/or the iron head based on the double-antibody sandwich principle, and the PLA-resistant kit provided by the invention is used for detecting the venom of the green bamboo and/or the iron head 2 Monoclonal and polyclonal antibodies to proteins with high affinity and strong specificity to Trimeresurus albolabris PLA 2 The ability of protein combination can realize rapid diagnosis of the bite of the green bamboo leaves or the iron heads, and greatly improve the snake bite detection efficiency and the accuracy of the detection result.
The invention provides a green bamboo PLA by combining the following examples 2 Protein-specific short peptide and anti-Trimeresurus albolabris PLA 2 Protein antibodies and snake bite detection kits are described in detail, but they are not to be construed as limiting the scope of the invention.
The term "anti-Trimeresurus PLA 2 Protein antibody, bamboo leaf green PLA 2 Antibodies and antibodies are used interchangeably and refer to a polypeptide capable of binding with phyllanthus urinaria PLA 2 Antibodies that bind specifically to proteins.
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 an antigen, also referred to as an "antigen-binding portion.
The term "isolated" refers to a substance that is separated from its original environment, i.e., the natural environment if it is a natural substance. If the naturally occurring polynucleotide and protein are not isolated or purified in vivo, the isolation of the polynucleotide or protein from the naturally occurring other materials is performed.
The term "hybridoma" refers to a cell obtained by fusing a myeloma cell and a B lymphocyte in the preparation of 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 carrier such as polystyrene, and an immune response is performed by utilizing the binding specificity of the antigen and antibody.
The invention provides an anti-bamboo leaf green PLA 2 Protein antibodies capable of binding to Trimeresurus albolabris PLA 2 Protein-specific binding and the antibodies are derived from the naturally purified Trimeresurus albolabris PLA 2 The protein is prepared.
Example 1
Downloading PLA from different snake sources from NCBI website 2 The protein sequences are compared by adopting comparison software (DNAMAN) to obtain comparison results (see figure 1), and the comparison results are obtained by comparing the green PLA of the bamboo leaves 2 The 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). Selecting the position of PLA by combining homology modeling analysis results 2 Polypeptide on protein surface to obtain green PLA 2 Protein candidate Polypeptide (PLA) 2 -peptide-1、PLA 2 -peptide-2 and PLA 2 -peptide-3), wherein PLA 2 The peptide-3 amino acid sequence is shown in SEQ ID NO. 1 (INLKLFCKKTSEQC).
Example 2
Green bamboo PLA 2 Protein-specific polypeptide antigen synthesis
Obtaining the green bamboo PLA by NCBI blast analysis and homology modeling analysis 2 Protein-specific polypeptide sequence PLA 2 Peptide-3 (see FIGS. 1 and 2). PLA (polylactic acid) 2 Peptide-3 antigen polypeptide was synthesized by solid phase synthesis from Jier Biochemical (Shanghai) Inc. Each short peptide antigen synthesizes 10mg of polypeptide antigen with the purity of more than 95 percent.
Example 3
PLA-resistant 2 Specific Polypeptide (PLA) 2 -peptide-3) polyclonal antibody preparation method comprising the steps of:
(1) Preparation of KLH-polypeptide composite antigen
A biochemical synthesis method is adopted to synthesize polypeptide and combine with hemocyanin (KLH) to prepare KLH-polypeptide complex antigen. The specific procedure was performed according to the instructions of the kit (K2039-5, bioVision, USA).
(2) Immunization of animals
The natural soldering bit KLH-polypeptide complex antigen was dissolved in physiological saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1 mg antigen) was emulsified with equal amounts of Freund's complete adjuvant (F-5881, sigma) and injected subcutaneously along the back of male New Zealand white rabbits at multiple sites, two rabbits per antigen injection. After a2 week interval, the boost was then injected intradermally into the back of male New Zealand white rabbits at multiple sites after emulsification with Freund's incomplete adjuvant (F-5506, sigma) and an equal volume of antigen (0.5 ml, containing 0.5mg antigen). Two boost immunizations were performed in total at 2 week intervals. Rabbit serum was collected 14 days after the second boost. The antigen is used as coating antigen, serum before immunization is used as negative control, and an indirect ELISA method is used for detecting the antibody titer. The method comprises the following specific steps:
1) Coating: KLH-polypeptide complex antigen (5. Mu.g/ml) was diluted with coating solution, and then added to 96-well plates in an amount of 100. Mu.l per well, and 100. Mu.l of coating solution was added to blank wells, and coated overnight at 4 ℃.
2) Washing: the coating liquid in the plate holes is poured off the next day, the plate holes are washed three times by using the washing liquid, each hole is 250 mu l, each time is 3-5 minutes, and the plate holes are patted dry as much as possible.
3) Closing: the blocking solution was 250. Mu.l per well at 37℃for 1h. And then washed three times and patted dry.
4) The positive wells were added with 100. Mu.l of gradient diluted rabbit immune serum, and the negative wells and blank wells were added with 100. Mu.l of blocking solution at 37℃for 1h. And then washed three times and patted dry.
5) 100 μl horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibody was added to the positive, negative and blank wells and incubated for 1h at 37deg.C in a dark incubator. And then washed three times and patted dry.
6) Color development: after washing, 100. Mu.l of the color-developing solution was added to each well, and incubated at 37℃for 15-30 minutes in the absence of light.
7) And (3) terminating: 100 μl of the stop solution was used for 450/630nm dual wavelength reading with a microplate reader.
The solution used in the above process is as follows:
1. coating liquid: 0.05M carbonate buffer at pH 9.6; 1.59g of sodium carbonate plus 2.93g of sodium bicarbonate were dissolved in 1L of deionized water.
2. Washing liquid: cells were buffered with Phosphate Buffered Saline (PBS) +0.1% Tween-20. And (3) injection: cell PBS: potassium chloride, 0.2g; potassium dihydrogen phosphate, 0.2g; sodium chloride, 8g; sodium dihydrogen phosphate 12, 2.16g and deionized water 1L.
3. Blocking solution (antibody dilution): the washing solution was 1g per 100 ml+bovine serum albumin (BSA).
4. Color development liquid: 3,3', 5' -Tetramethylbenzidine (TMB) single component color development liquid (Solarbio, beijing).
5. Stop solution: 2mol/L sulfuric acid, 178.3ml water+21.7 ml concentrated sulfuric acid, and the mixture was stirred slowly and mixed well.
Example 4
The separation and purification method of the green bamboo leaf PLA2 protein comprises the following steps:
a Resource S column is arranged on an FPLC (fast protein liquid chromatography, AKTA pure) instrument, 50mg of the thick bamboo leaf green toxin is weighed and dissolved in 2ml A liquid after a column bed is washed according to instructions, and the thick bamboo leaf green toxin is arranged on the balanced Resource S column from a sample adding valve. After detection by ultraviolet detector at 280nm and 215nm, collecting each peak, collecting each eluting peak respectively, freeze-drying and storing at-20deg.C for use. The method comprises the following specific steps: equilibrated with solution A (flow rate 3 ml/min), loaded, unbound protein washed off with solution A, and eluted linearly with solution B, and the peaks were collected. The solution used in the above process is as follows:
liquid A (KCl: 0.2g, KH) 2 PO 4 ：0.2g，Na 2 HPO 4 ·12H 2 O3.47 g, pH 7.2); liquid B (KCl: 0.2g, KH) 2 PO 4 ：0.2g，Na 2 HPO 4 ·12H 2 O:3.47g, naCl:58g, pH 7.2) was filtered through a 0.45 μm filter (Millipore) and subjected to ultrasonic degassing.
With anti-PLA 2 WB detection is carried out on peptide-3 polypeptide antigen antibody so as to confirm the position of target proteinPeaks (see FIG. 3). Finally, carrying out modified SDS-PAGE electrophoresis to detect protein purification on the purified Trimeresurus albolabris PLA2 protein, and the result shows that the natural Trimeresurus albolabris PLA2 protein is obtained by separating and purifying from snake venom respectively (see figure 4).
Example 5
Green bamboo PLA 2 Preparation method of protein monoclonal antibody
1) Natural bamboo leaf green PLA 2 The protein antigen immunizes mice and spleen cells of the immunized mice are obtained.
Natural Trimeresurus albolabris PLA prepared by example 3 2 The protein was immunized against 5 Balb/c mice. Wherein, balb/c mice are white variety laboratory mice, each of which is subjected to at least 4 immunizations and 3 blood sampling tests on average, and target mice with higher titers (selected from the above natural antigens) are selected after the first, second and third blood sampling tests, in this example, 2 mice are selected from 5 mice, and spleen cells of these 2 mice are obtained.
2) Fusing spleen cells and mouse myeloma cells, and screening out PLA which can be matched with natural green bamboo leaves 2 A hybridoma cell line that binds to a protein antigen.
Carrying out cell fusion on SP2/0 mouse myeloma cells from Balb/c mice and spleen cells of 2 screened mice, culturing, observing, detecting and carrying out yin-yang contrast test after fusion, thus obtaining the anti-Trimeresurus albolabris PLA 2 Hybridoma cell lines of the protein antibodies are cultured and selected continuously.
3) Inoculating hybridoma cells into abdominal cavity of a mouse for induction culture, culturing to obtain a cell culture solution, and purifying from the cell culture solution to obtain the cell culture solution which can be used with Trimeresurus albolabris PLA 2 anti-Trimeresurus albolabris PLA immunoreactive with protein antigen 2 Protein monoclonal antibodies.
Before inoculating hybridoma, injecting 0.5 mL/mouse into the abdominal cavity, and injecting 2×10 hybridoma cell suspension prepared by the method into the abdominal cavity after one week 6 Per mL, each mouse was injected with 0.5mL hybridoma cell suspension.
Collecting abdomen under aseptic condition about 7-10 days after inoculating hybridomaCentrifuging 12000g ascites of obviously swelled mice for 10min to remove lipid, cell and other impurities, collecting supernatant obtained by centrifuging, and inactivating at 56 deg.C for 30min to obtain PLA 2 Protein monoclonal antibodies.
Detection of Trimeresurus albolabris PLA by indirect ELISA (Enzyme Linked ImmunosorbentAssay, enzyme-linked immunosorbent assay) 2 Protein antibody titers (coating antigen is natural antigen). Specifically, five natural snake venom and natural PLA are used 2 Screening monoclonal antibody by using protein as coating antigen, wherein five natural snake venom antigens are shown as A in figure 5, 100 mu l each hole is coated with snake venom antigen with concentration of 10 mu g/ml on 96-well plate, BSA is blocked and washed, the monoclonal antibody is diluted by antibody secreted by the prepared hybridoma cell strain, and incubated, after washing, anti-mouse HRP labeled secondary antibody is added for incubation, and after washing, display and OD are carried out 450 And (5) reading.
The specific results are shown in FIG. 5. Only the Trimeresurus venom can carry out specific affinity reaction with the monoclonal antibody, and the monoclonal antibodies secreted by different kinds of hybridoma cell strains can be matched with the natural PLA 2 The proteins undergo an affinity reaction.
PLA is used as the material 2 And (3) screening monoclonal cell lines and antibodies with high antibody titers by taking peptide-3 polypeptides as coating antigens. With PLA 2 Second round screening of polyclonal antibodies for PLA by peptide-3 2- The peptide-3 antigen (100 mu l per well, concentration 10 mu g/ml) is coated on a 96-well plate, BSA is blocked and washed, diluted monoclonal antibody is added for incubation, anti-mouse HRP labeled secondary antibody is added for incubation after washing, and display and OD are carried out after washing 450 And (5) reading.
The results show that PLA 2 -mc8、PLA 2 -mc11 and PLA 2 -mc14 is capable of associating with PLA 2 Peptide-3 polypeptide antigen binding (FIG. 6). Monoclonal antibody PLA 2 -mc8 delegated the determination of the light chain variable region and the heavy chain variable region by su Jin Weizhi biotechnology limited, specific sequences are shown in SEQ ID No. 2 and SEQ ID No. 3. Monoclonal antibody PLA 2 The results of the analysis of the mc8 variable regions are shown in Table 1.
Table 1 monoclonal antibody PLA 2 -sequence of regions of the mc8 variable region
Figure BDA0003301311960000101
It can be seen that the monoclonal antibody is prepared by the in-vivo induction method of the abdominal cavity inoculated animal body in the embodiment, after the cloning of the hybridoma cells is successful, the hybridoma is inoculated into the abdominal cavity of the mouse to induce a large amount of ascites, and simultaneously a large amount of antibody is secreted into the ascites, so that the concentration of the antibody is higher, and the bamboo leaf green PLA with higher titer can be conveniently screened out 2 Protein antibodies.
Example 6
Anti-natural bamboo leaf green PLA 2 The preparation method of the protein polyclonal antibody comprises the following specific steps:
natural bamboo leaf green PLA 2 The proteins were dissolved in physiological saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1 mg antigen) was emulsified with equal amounts of Freund's complete adjuvant (F-5881, sigma) and injected subcutaneously along the back of male New Zealand white rabbits at multiple sites, two rabbits per antigen injection. After a2 week interval, the boost was then injected intradermally into the back of male New Zealand white rabbits at multiple sites after emulsification with Freund's incomplete adjuvant (F-5506, sigma) and an equal volume of antigen (0.5 ml, containing 0.5mg antigen). Two boost immunizations were performed in total at 2 week intervals. Rabbit serum was collected 14 days after the second boost.
Example 7
Anti-bamboo leaf green PLA 2 Protein monoclonal antibody and anti-Trimeresurus albolabris PLA 2 The protein polyclonal antibody is respectively combined with the Trimeresurus albolabris PLA2 protein antigen and five snake venom (Trimeresurus albolabris, viper and soldering iron) to detect, and the specific steps are the same as the step of detecting the antibody titer by the indirect ELISA method in the embodiment 5. Simultaneously, the specificity and the space accessibility of the antibody are detected by a western blot method (WB).
The results are shown in FIG. 7. Wherein A and B are each anti-PLA 2 Monoclonal antibody PLA 2 -mc8 and anti-PLA 2 ELISA and WB detection results of polyclonal antigens. ELISA and WB results showed anti-PLA 2 Monoclonal antibody PLA 2 -mc8 and anti-PLA 2 Polyclonal antibodiesAll have good specificity.
Example 8
Monoclonal antibody and polyclonal antibody pairing detection
The specific method of antibody pairing is as follows. Coating: the anti-natural PLA2 polyclonal antibody is diluted into 10 mug/ml by coating liquid, 100 mug/well is coated overnight at 4 ℃; closing: removing the coating liquid, washing with PBST for 3 times, 5min each time, and incubating 200 μl of blocking liquid per well at 37deg.C for 1 hr; incubating the sample: the blocking solution was discarded, PBST was washed 3 times, 5min each time, and five crude toxins (green bamboo leaves, viper, cobra and iron head) were diluted in PBS at concentrations of 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, 7.8125ng/ml, and 100. Mu.l were added per well. Incubating for 1h at 37 ℃; incubating PLA2 monoclonal antibodies: the samples were discarded and PBST washed 3 times for 5min each, with HRP-bearing monoclonal antibody dilution ratio of 1:3000, 100 μl was added per well and incubated for 1h at 37deg.C; developing: discarding monoclonal antibody, washing with PBST for 3 times, adding 100 μl TMB color developing solution, and incubating at 37deg.C in dark place for 10min; and (3) terminating: 100 μl of stop solution was added to each well, and the absorbance at 450nm was determined for each well.
The results are shown in FIG. 8. The results showed that the pairing mode with polyclonal antibody as coating antibody and monoclonal antibody as detection antibody was the optimal pairing mode.
Example 9
Method for establishing standard curve of Trimeresurus albolabris venom by double-antibody sandwich ELISA method
Anti-greenvenom PLA after PBS dilution 2 Protein polyclonal antibody (10. Mu.g/ml) was added to 96-well plates at 100. Mu.l per well and blank wells were coated with 100. Mu.l coating solution at 37℃for 1 hour; pouring out the coating liquid in the plate holes, washing with the washing liquid for three times, and taking 250 mu l of each hole for 5 minutes each time and taking the paste as dry as possible; sealing each hole with 250 μl of sealing solution at 37deg.C for 1 hr, washing for three times, and drying; adding 100 μl of diluted snake venom into the positive well, adding 100 μl of blocking solution into the negative well and blank well, incubating at 37deg.C for 1 hr, washing for three times, and drying; positive, negative and blank wells were filled with 100. Mu.l horseradish peroxidase (HRP) -labeled anti-Green snake venom PLA 2 Protein (concentration of antibody stock solution is 1mg/ml, diluted 1:3000), and is protected from light at 37 DEG CIncubating for 1h in an incubator, washing for three times and drying; after washing, adding a color development solution into 100 mu l of each hole, and incubating for 25 minutes at 37 ℃ in a dark place; 100 μl of the stop solution was used for 450/630nm dual wavelength reading with a microplate reader.
The standard curve of the Trimeresurus venom detection is shown in FIG. 9. The regression equation is y=0.0131x+0.1784, where R 2 ＝0.996。
Example 10
A kit for detecting the venom of Trimeresurus albolabris by double-antibody sandwich ELISA comprises the following components:
coated with anti-bamboo leaf green PLA 2 A solid phase carrier of the protein polyclonal antibody; the coating concentration is 10 mug/ml;
horseradish peroxidase marked anti-bamboo leaf green PLA 2 A protein monoclonal antibody;
diluting solution, chromogenic substrate, stop solution, sealing film and positive standard substance, wherein the positive standard substance is natural PLA of folium Bambusae 2 The preparation method of the protein solution comprises the following steps:
1. preparing a crude toxic solution, specifically, respectively taking out a proper amount of laboratory-preserved bamboo leaf green freeze-dried powder, and dissolving with a proper amount of ultrapure water to obtain the crude toxic solution.
2. The crude toxic solution sample was diluted 5-fold with phosphate buffer, which was the same as above (solution A). Then the mixture is filtered by a 0.22 mu m filter membrane and loaded on a Resource S column, and the mixture is eluted by a linear gradient of 0 to 1mol/L NaCl solution. Then, each elution peak was detected and collected at 280nm and 215nm wavelengths and lyophilized until a natural PLA2 protein standard with a purity of greater than 95% was obtained.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Kunming animal institute of China academy of sciences
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Claims (8)

1. Green bamboo PLA 2 The protein specific short peptide is characterized in that the amino acid sequence of the short peptide is shown as SEQ ID NO. 1.
2. An anti-Trimeresurus albolabris PLA screened from the short peptide of claim 1 2 Protein monoclonal antibody PLA 2 -mc8, characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as set forth in SEQ ID NO:2, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO:3.
3. ELISA detection kit for snake bite of green bamboo leaves is characterized by comprising coating anti-green bamboo leaves PLA 2 Solid phase carrier of polyclonal antibody of protein, enzyme-labeled anti-Trimeresurus albolabris PLA as defined in claim 2 2 Protein monoclonal antibody PLA 2 -mc8。
4. The test kit of claim 3, wherein the anti-bambusa PLA 2 The protein polyclonal antibody is PLA separated and purified from Trimeresurus chinensis venom 2 The protein immune animal is prepared.
5. The test kit of claim 4, wherein the bambusa aculeata PLA 2 The amino acid sequence of the protein is shown in SEQ ID NO: 4.
6. The detection kit according to claim 3, wherein the solid phase carrier is resistant to Trimeresurus chinensis PLA 2 The coating concentration of the polyclonal antibody of the protein is 1-100 mug/ml.
7. The test kit according to any one of claims 3 to 6, further comprising a washing solution, a sample dilution solution, a substrate, a color-developing solution and a positive standard.
8. The test of claim 7The test kit is characterized in that the positive standard comprises separated and purified PLA 2 Protein or the green bamboo PLA of claim 1 2 Protein-specific short peptides.
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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
CN110684105A (en) * 2019-09-11 2020-01-14 江苏莱森生物科技研究院有限公司 anti-HSP 90 monoclonal antibody and kit
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