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Brown planthopper VgR polypeptide and multi-resistant preparation method thereof

A technology for antibody preparation and anti-BPH, applied in chemical instruments and methods, specific peptides, anti-animal/human immunoglobulins, etc., can solve the problems of no commercial products and limited in-depth research on the biological functions of insect VgR proteins

Inactive Publication Date: 2015-08-26
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there are currently no commercial products of antibodies against VgR proteins of insects such as brown planthopper on the market, which limits the in-depth study of the biological functions of insect VgR proteins

Method used

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  • Brown planthopper VgR polypeptide and multi-resistant preparation method thereof
  • Brown planthopper VgR polypeptide and multi-resistant preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Analysis of the VgR sequence of the brown planthopper and the design and synthesis of the VgR polypeptide

[0016] According to the N. lugens VgR sequence on GenBank (accession number: ADE34166), it is known that the VgR protein sequence of N. lugens contains 1931 amino acids. Using the Protean program module in the DNAstar software to analyze the characteristics of the N. lugens VgR protein, it is known that the molecular weight of the protein is 215198.74 dahl Dayton, the isoelectric point is 4.95, which is an acidic protein. After further analyzing the characteristics of the protein's amino acid sequence, such as antigenicity, hydrophilicity, and surface possibility, a polypeptide sequence with the sequence RKGNADQSVATKSD was screened out and suitable for use as an antigen (1295aa—1308aa ). In order to facilitate cross-linking with the carrier protein and increase the immunogenicity of the polypeptide, a cysteine ​​C was added to the N-terminus of the abov...

Embodiment 2

[0017] Example 2: Cross-linking of polypeptides and carrier proteins

[0018] Use MBS as a cross-linking agent to cross-link the carrier protein KLH with the synthetic polypeptide: dissolve KLH with cross-linking buffer to a concentration of 10 mg / mL; dissolve MBS in DMF to a concentration of 10 mg / mL; dissolve the dissolved KLH solution and MBS solution by Mix at a ratio of 10:1 (W / W), activate KLH at room temperature for 30 minutes; purify the activated KLH solution with Sephadex G‐25; mix the activated KLH solution with the peptide solution at a ratio of 1:1 (W / W), at room temperature The reaction was carried out for 3 hours; the above reaction solution was dialyzed in PBS at 4° C. overnight to obtain the polypeptide-KLH complex protein.

Embodiment 3

[0019] Embodiment three: the immunization of experimental animal

[0020] New Zealand male rabbits of appropriate age were selected as immunized animals, and 2-3 mL of blood was collected from the ear vein before immunization, which was used as a negative control for subsequent ELISA detection. For the first immunization, 0.5 mg of polypeptide-KLH complex protein was dissolved in 0.5 mL of PBS solution, fully mixed with an equal volume of Freund's complete adjuvant to emulsify, and injected subcutaneously into rabbits at multiple points. Two weeks later, the first booster immunization was carried out. Dissolve 0.5 mg of polypeptide-KLH cross-linked complex protein in 0.5 mL of PBS solution, fully mix and emulsify with an equal volume of Freund's incomplete adjuvant, and inject subcutaneously at multiple points. The booster immunization with the same operation was carried out every 3 weeks, a total of 3 times before and after. One week after each booster immunization, a small ...

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Abstract

The invention discloses a brown planthopper VgR polypeptide and multi-resistant preparation method thereof. The amino acid sequence of VRG polypeptide is RKGNADQSVATKSD, the preparation method for polyclonal antibody comprises the following steps: (1) performing epitope analysis on brown planthopper VRG protein antigen; (2) designing and analyzing brown planthopper VgR; (3) crosslinking synthetic peptide and carrier protein; (4) preparing rabbit antibody brown planthopper VgR polypeptide antibody; (5) collecting, separating to obtain serum containing antibody, purifying antibody to obtain the antibody of brown planthopper VgR polypeptide. The brown planthopper VgR polypeptide polyclonal antibody is good in specificity, and high in purity, has specific binding reaction with the brown planthopper VgR protein and is used for performing correlation research for brown planthopper VgR protein, such as analysis of character, function, expression profile and content.

Description

technical field [0001] The invention relates to a preparation method of a polypeptide and an antibody thereof. The antibody is mainly used for detecting natural protein antigens, specifically a preparation method of a rabbit polyclonal antibody against brown planthopper VgR polypeptide. Background technique [0002] The full name of VgR is vitellogenin receptor, which is the vitellogenin receptor and is the main receptor that mediates the endocytosis of insect vitellogenin. It belongs to the low-density lipoprotein family. VgR plays an important role in reproductive processes such as oogenesis. After searching, there are no commercial products of antibodies against VgR proteins of insects such as brown planthopper in the market, which limits the in-depth study of the biological functions of insect VgR proteins. Contents of the invention [0003] (1) The purpose of the present invention is to provide a VgR polypeptide whose sequence is: RKGNADQSVATKSD. [0004] (2) Anothe...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K16/28
CPCC07K14/705C07K16/28
Inventor 俞晓平许益鹏申屠旭萍刘光富郝培应杨倩倩
Owner CHINA JILIANG UNIV
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