CN113512106B - Synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof - Google Patents
Synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof Download PDFInfo
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- CN113512106B CN113512106B CN202110465269.7A CN202110465269A CN113512106B CN 113512106 B CN113512106 B CN 113512106B CN 202110465269 A CN202110465269 A CN 202110465269A CN 113512106 B CN113512106 B CN 113512106B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
Abstract
A synthetic peptide of grass carp C5aR protein and its polyclonal antibody are characterized by that it utilizes DNA star software to comprehensively analyze C5aR sequence, and utilizes Fmoc method to chemically synthesize the polypeptide shown by SEQ ID No.2 containing 2-20 th amino acids from N end, and then makes the polypeptide undergo the process of purification, and couples the polypeptide sulfhydryl group on the carrier protein KLH to form C5aR-KLH conjugate as polypeptide antigen, and makes it pass through several times of immunization of experimental-grade Japanese white rabbit to obtain the concentrated synthetic peptide polyclonal antibody of grass carp C5aR protein. The concentrated grass carp C5aR polyclonal antibody meeting the subsequent experimental requirements can be obtained by the method, can be used for grass carp C5aR protein expression detection, and lays an important material foundation for the research of C5aR protein functions and action mechanisms.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to synthetic peptide of grass carp C5aR protein, and a polyclonal antibody prepared by taking a conjugate obtained by coupling the synthetic peptide with carrier protein KLH as an antigen.
Background
C5aR is a G protein-coupled receptor, also known as CD88, which is one member of the family of G protein-coupled receptors with 7 transmembrane domains, expressed naturally in the membranes of a variety of cells, mainly on neutrophils, mast cells and macrophages. Its natural expression form has close relation with its mediated biological function. C5aR plays an important role in tissue inflammation, and the mediation of binding of C5aR to its ligand leads to an increase in intracellular calcium ions and an intracellular signaling cascade, thereby exerting various biological effects, such as: stimulating cells of myeloid origin such as neutrophils and monocytes to release histamine and lysosomal enzymes, inducing smooth muscle contraction, enhancing vascular permeability, mediating the aggregation of inflammatory cells such as neutrophils and monocytes to inflammatory sites, promoting endothelial cell expression of adhesion molecules, and immunomodulating by inducing or inhibiting the release of monocyte cytokines.
Grass carp (A)Ctenopharyngodonidella) Is the freshwater fish culture breed with the largest yield in China, and Grass carp reovirus (Grass carp re)GCRV) seriously restricts the healthy culture of grass carps. Inflammatory factors are abundantly expressed in grass carp hemorrhage, but the inflammatory mechanism regulated by C5aR and its ligand in grass carp is not clear, and the biological function exerted by C5aR in grass carp is not clear. In view of the research defect of C5aR in grass carp hemorrhagic disease, the synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof are obtained, which has great significance for further discussing the biological function of C5aR and developing and applying.
Disclosure of Invention
The invention aims to solve the technical problem that the experimental materials in the prior art are deficient, and provides the grass carp C5aR protein synthetic peptide and the polyclonal antibody thereof, and the grass carp C5aR protein synthetic peptide polyclonal antibody which meets the subsequent detection requirements is constructed by analyzing the grass carp C5aR protein sequence by using the method, so that an important material foundation is laid for the research of the functions and action mechanisms of the C5aR protein.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a synthetic peptide of grass carp C5aR protein has an amino acid sequence shown in SEQ ID NO. 2.
The invention also provides an antigen which is obtained by coupling the synthetic peptide of the grass carp C5aR protein and a carrier protein KLH.
The invention also provides a polyclonal antibody which is obtained by immunizing animals with the antigen.
The specific preparation steps of the above-mentioned polyclonal antibody are as follows:
(1) according to the grass carp C5aR protein complete sequence shown in SEQ ID NO.1, comprehensively analyzing the characteristics of the C5aR sequence by using DNA star software, and determining the grass carp C5aR protein synthetic peptide sequence shown in SEQ ID NO. 2;
(2) coupling grass carp C5aR protein synthetic peptide with carrier protein KLH to obtain antigen;
(3) animals are immunized with an antigen.
The synthetic peptide sequence of the grass carp C5aR protein in the step (1) is the amino acid at the 2 nd to 20 th positions of the outside region of the full sequence of the grass carp C5aR protein shown in SEQ ID NO. 1.
The animal in the step (3) is an experimental-grade Japanese big ear white rabbit.
The invention has the beneficial effects that: the obtained concentrated grass carp C5aR polyclonal antibody meeting the requirements of subsequent experiments can be used for grass carp C5aR protein expression detection, and lays an important material foundation for the research of C5aR protein functions and action mechanisms.
Drawings
FIG. 1 is a graph showing the result of epitope prediction analysis of grass carp C5aR protein according to the present invention;
shows that: the target protein is a seven-transmembrane protein, the expression difficulty is extremely high, the antigen is not suitable for preparing an expression route, and the optimal mode of preparing the antigen by selecting synthetic polypeptide is selected.
FIG. 2 is a graph showing the results of analysis of the transmembrane domain of the grass carp C5aR protein of the present invention;
shows that: the 2-20aa section is the N end of the protein and is positioned in the outside area, so that the exposition property is better, the specificity, the immunogenicity and the hydrophilicity are better, and the preparation method is suitable for preparing the synthetic peptide antibody of the C5aR protein.
FIG. 3 is antiserum ELISA test data;
wherein: the polypeptide coating amount is 100ng, and the antibody dilution ratio is 1:1000,1: 5000,1: 10000,1: 50000,1: 100000,1: 200000.
FIG. 4 shows the WB detection results of the antigen of the present invention;
wherein: the polypeptide coating amount is 100ng, and the antibody dilution ratio is 1:1000,1: 5000,1: 10000,1: 50000,1: 100000,1: 200000.
FIG. 5 is a graph showing the Western Blot hybridization results of different organs or tissues of a healthy grass carp using the polyclonal antibody against grass carp C5aR of the present invention.
FIG. 6 shows the Western Blot detection result of C5aR expression in liver of grass carp with hemorrhage using the polyclonal antibody to grass carp C5aR obtained by the present invention;
wherein: beta-actin is internal reference protein; A. b, C, D, E, F represent the onset, 12 hours, stage 1, stage 3, stage 7 and stage 10 of grass carp infection with leukemia virus, respectively; stage 1 indicates onset, stage 3 indicates onset into severe stage, stage 7 indicates onset into recovery stage, and stage 10 indicates recovery.
Detailed Description
The following examples are further illustrative and explanatory of the invention, and are not restrictive thereof.
Example 1 design of synthetic peptide of C5aR protein
The grass carp C5aR protein sequence shown in SEQ ID NO.1 is searched from an NCBI website (https:// www.ncbi.nlm.nih.gov /) GenBank database, the characteristics of the C5aR sequence are comprehensively analyzed by using DNA star software according to the aspects of 4 parameters, specificity, synthesis difficulty and the like of an antigen index, a hydrophilic structure, a flexible region and a surface probability structure, the synthesized peptide sequence is determined to contain 2-20 amino acids from the N end, the sequence is shown in SEQ ID NO.2, and please refer to the graph 1 and the graph 2 in combination.
Example 2 Synthesis of N-terminal polypeptide of C5aR protein and antigen
The C5aR protein N-terminal polypeptide is synthesized by Liberty full-automatic polypeptide synthesizer of CME company in USA. The chemical synthesis adopts Fmoc method, and the synthesis purity is 98 percent. The synthesized polypeptide (C5 aR protein synthetic peptide) is dissolved in 8M urea-PBS buffer solution, and sulfhydryl is coupled to carrier protein KLH to form C5aR-KLH conjugate as antigen for preparing polyclonal antibody.
EXAMPLE 3 preparation of polyclonal antibody against synthetic peptide of C5aR protein
For the first immunization, 350 mu g of C5aR-KLH conjugate is taken and fully emulsified with an equal amount of complete Freund's adjuvant, and then injected into the neck and back of a Japanese big-ear white rabbit at multiple subcutaneous injection points, wherein each injection point is about 100 mu g; a second immunization 12 days later, using incomplete Freund's adjuvant in combination with C5aR-KLH conjugate at a dose of 0.35 mg; a third immunization 26 days after the first immunization, using incomplete Freund's adjuvant in combination with C5aR-KLH conjugate at a dose of 0.35 mg; the fourth immunization was carried out 40 days after the first immunization, and incomplete Freund's adjuvant was mixed with C5aR-KLH conjugate at a dose of 0.35 mg; a fifth immunization 54 days after the first immunization was performed using incomplete Freund's adjuvant in a 0.35 mg dose in combination with C5aR-KLH conjugate. The blood collection time for immunizing Japanese white rabbits with big ears is 66 days after the first immunization, blood is collected from carotid arteries of the Japanese white rabbits with big ears, and after serum is separated, the blood is stored in an ultra-low temperature refrigerator (Thermo Fisher Scientific, USA) for later use. The antiserum ELISA test was performed on the separated serum, and the results showed that the antiserum could be detected by ELISA after dilution at 1:1000, 1:5000 and 1:10000, as shown in FIG. 3.
Example 4 affinity purification of antibody sera
Weighing 10mg of the synthesized polypeptide, dissolving with 2ml of coupling Buffer (50 mM tris, 5Mm EDTA-NA), placing 2ml of sulfolinking gel (Thermo, USA) in a chromatographic column, adding the antigen into the chromatographic column, rotating for 3 hours on a rotary incubator, standing on a chromatographic rack for 30 minutes, and connecting a constant flow pump. 40ml 1 × PBS flow rate 2.0, 30ml 1 × Gly, 30ml 1 × PBS column environment balance to neutral; 1/20M sodium chloride is added into the sample, the flow rate is 1.5, and the sample is subjected to column chromatography twice; 40ml Wash buffer (1 XPBS 320mM NaCl) was added and 1 XPY flow 0.6 was added and samples were collected. After collection, the antibody was eluted by adding an excess of acid, and then the collected antibody was equilibrated to neutral with 1 × PBS, and put into a dialysis bag, after mixing, about 30 μ l of the collected antibody was used for antibody concentration measurement, and the remaining antibody was dialyzed and concentrated in 1 × PBS50% glycerol overnight. Concentrated grass carp C5aR polyclonal antibodies with the concentrations of 1.29 mg/ml and 1.83 mg/ml are obtained respectively, and the results of antigen western blot show that the antibodies can be detected under the conditions of dilution of 1:1000, 1:5000 and 1:10000, and the combination is shown in figure 4.
Example 5 Western blot analysis of polyclonal antibodies
In order to verify whether the obtained grass carp C5aR protein synthetic peptide polyclonal antibody can be used for a subsequent Western Blot experiment, the obtained grass carp C5aR protein synthetic peptide polyclonal antibody is used for detecting the expression conditions of grass carp C5aR in the liver, spleen, kidney, head kidney, intestine, gill and muscle of a healthy grass carp and the expression conditions of grass carp at different disease onset time points in the liver of the grass carp with a disease, and three grass carp samples are collected in each group of experiments. Firstly, 0.1 g of tissues are respectively sheared from each detected organ or tissue, added into a homogenate tube filled with 1 ml of RIPA lysate, 10 mul of protease inhibitor and 2 sterile steel balls, ground by using a freezing mixing ball mill with the homogenizing speed of 55 Hz and 1650r/s, then cracked for 10 minutes after grinding, and then centrifuged at 4 ℃ and 12000 rpm for 15 minutes, and the centrifuged supernatant is respectively transferred into 1.5 ml centrifuge tubes for subsequent experiments. According to the total protein amount of each sample, 20 mu g of the sample loading amount is spotted into PAGE gel with the separation gel concentration of 15% by 100V electrophoresis until bromophenol blue reaches the bottom of the gel. Then the grass carp anaphylactic toxin C5a protein (8 kD) is subjected to membrane transfer. Wash 3 times with 1 XPBST buffer, shake 10 min per destaining. Adding appropriate amount of sealing solution, and sealing on decolorizing shaking table for 15 min. The membrane was added to a multi-well box, diluted with polyclonal antibody to grass carp anaphylactotoxin C5a (diluted 1:1000 with blocking solution), and incubated with the antibody at 4 ℃ overnight. After the incubation was completed, the cells were washed 3 times with 1 XPBST buffer for 10 min each. Dilutions of HRP-labeled secondary antibody (diluted 1: 2000 with blocking solution) were incubated with the membrane for 60 min, followed by 3 washes with 1 XPBST buffer for 10 min each. And finally mixing the chromogenic solution A and the chromogenic solution B (Thermo) in equal volume, keeping out of the sun, adding 200 mu L of each membrane, and standing for 1-2 min. The film was placed into a fluorescence imager, observed and photographed. The results show that the expression result of grass carp C5aR in different tissues of healthy grass carps is shown in FIG. 5, and the expression of the protein of C5aR in different tissues is obviously different, wherein the protein of C5aR is mainly expressed in liver and is significantly different from other six tissues (P < 0.05). The expression results of grass carp C5aR in grass carp liver with hemorrhage at different onset time points are shown in FIG. 6, and after GCRV infection, the protein expression of C5aR shows a step-like and continuous increase in grass carp liver. The initial infection stage and the latent stage are at the same level, the disease stage and the death stage are at the same level, and the recovery stage are at the same level. The above results demonstrate that the method described in the present invention can obtain the concentrated grass carp C5aR protein synthetic peptide polyclonal antibody for grass carp C5aR expression detection.
Sequence listing
<110> Hunan agriculture university
<120> synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof
<130> 0003
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 358
<212> PRT
<213> Synthesis ()
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Met Glu Gly Asp Tyr Leu Asp Tyr Gly Pro Phe Asp Phe Ser Asn Glu
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Ser Tyr Ser Cys Glu Asp Pro Glu Asn Cys Thr Asp Ile Glu Phe Pro
20 25 30
Asn Glu Ile Leu Met Ser Glu Ile Gly Leu Arg His Trp Ile Ala Leu
35 40 45
Val Cys Tyr Ser Ile Val Phe Leu Leu Gly Val Pro Gly Asn Gly Leu
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Val Val Trp Val Thr Ala Phe Arg Met Pro Asn Ser Val Asn Ala Gln
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Trp Phe Leu Asn Leu Ala Ile Ala Asp Leu Leu Cys Cys Leu Ser Leu
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Pro Phe Leu Met Val Ser Leu Ala Gln Asp Lys His Trp Pro Phe Gly
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Asp Leu Ala Cys Lys Leu Leu Ser Gly Leu Phe Tyr Met Met Met Tyr
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Cys Ser Val Leu Leu Leu Val Val Ile Ser Leu Asp Arg Phe Leu Leu
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Val Thr Lys Pro Val Trp Cys Gln Asn His Arg His Pro Arg Gln Ala
145 150 155 160
Arg Cys Val Cys Phe Phe Val Trp Ile Leu Ala Leu Leu Gly Gly Ser
165 170 175
Pro Gln Phe Ala His Met Glu Thr Arg Arg Asp Glu Thr Lys Thr Leu
180 185 190
Cys Asn Ser Ala Tyr Asn Ser Phe Gly His Ala Trp Ala Val Thr Leu
195 200 205
Thr Arg Ser Phe Leu Ser Phe Leu Leu Pro Phe Leu Ile Ile Cys Gly
210 215 220
Ser His Trp Met Val Tyr Arg Thr Met Ser Ser Gly Arg Gly Gln Arg
225 230 235 240
Arg Ser Asp Lys Ser Ala Arg Thr Leu Arg Val Ile Leu Ala Leu Val
245 250 255
Leu Ser Phe Phe Leu Cys Trp Ser Pro Leu Gln Ile Val Asp Leu Leu
260 265 270
Glu Leu Ile Leu Tyr Glu Gln Ser Glu Ser Leu Arg Ala Asn Val Arg
275 280 285
Leu Ala His Val Leu Thr Leu Cys Leu Ala Tyr Ile Asn Ser Cys Leu
290 295 300
Asn Pro Leu Leu Tyr Val Cys Leu Gly Arg Gly Phe Lys Lys Asn Leu
305 310 315 320
Val Ser Ser Leu Arg Ser Val Leu His Phe Ala Ser Glu Ala Pro Thr
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Asn Arg Thr Ser Met Thr Ala Asn Ser Lys Ser Thr Thr Asp Gly Met
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Phe Arg Glu Lys Pro Val
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Claims (5)
1. A synthetic peptide of grass carp C5aR protein has an amino acid sequence shown in SEQ ID NO. 2.
2. An antigen obtained by coupling the synthetic peptide of grass carp C5aR protein of claim 1 with a carrier protein KLH.
3. A polyclonal antibody, which is obtained by immunizing an animal with the antigen of claim 2.
4. The polyclonal antibody of claim 3, which is prepared by the following steps:
(1) according to the grass carp C5aR protein complete sequence shown in SEQ ID NO.1, comprehensively analyzing the characteristics of the C5aR sequence by using DNA star software, and determining the grass carp C5aR protein synthetic peptide sequence shown in SEQ ID NO. 2;
(2) coupling grass carp C5aR protein synthetic peptide with carrier protein KLH to obtain antigen;
(3) animals are immunized with an antigen.
5. The polyclonal antibody of claim 4, wherein the animal in step (3) is an experimental grade Japanese big ear white rabbit.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5480974A (en) * | 1993-06-18 | 1996-01-02 | The Scripps Research Institute | Antibodies to human C5a receptor |
| CN106957366B (en) * | 2016-01-12 | 2022-02-01 | 上海昀怡健康科技发展有限公司 | C5aR antibody and preparation method and application thereof |
| CN109651497B (en) * | 2018-12-29 | 2021-03-19 | 新乡医学院 | Grass carp FoxO1 polypeptide antigen and antibody, and preparation method and application thereof |
| CN110218250B (en) * | 2019-06-14 | 2020-06-16 | 湖南农业大学 | Preparation method of grass carp complement component 5 polyclonal antibody |
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