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Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide

A silk and protein technology, applied in the direction of serum immunoglobulin, anti-animal/human immunoglobulin, etc., to achieve high sensitivity and strong specificity

Inactive Publication Date: 2014-01-15
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current advanced technology for protein detection is based on the antibody-antigen Western Blotting or ELISA method, but the preparation of antibodies often uses natural protein macromolecules, so it cannot meet the above research requirements

Method used

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  • Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide
  • Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide
  • Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide

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Embodiment Construction

[0014] Download Bombyx mori ( Bombyx mori ) The amino acid sequence of the heavy chain (H-chain) of silk fibroin (as shown in SEQ No.1). Through statistical analysis, it is determined that the "GYGAGAGAGYGA" polypeptide fragment has a high frequency of occurrence, a total of 47, which is the characteristic polypeptide of silkworm silk fibroin, so this fragment is synthesized as an antigen to prepare antibodies.

[0015] The preparation method of the present invention will be specifically described below.

[0016] Synthesis of complete antigen

[0017] Use a polypeptide synthesizer to synthesize a hapten (cysteine) to couple the polypeptide to KLH. Through the action of a cross-linking agent, the sulfhydryl group on the cysteine ​​at the N-terminal of the "CGYGAGAGAGYGA" polypeptide forms a covalent bond with the KLH primary amine, Thus, the polypeptide and KLH are coupled to obtain the complete antigen.

[0018] Dilute the complete antigen with an appropriate amount of no...

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Abstract

The invention discloses a method of preparing a bombyx mori silk fibroin specific antibody by utilizing a characteristic dodecapeptide. The method comprises the following steps: synthesizing a polypeptide with a "CGYGAGAGAGYGA" sequence, coupling the polypeptide with keyhole limpet hemocyanin (KLH) so as to obtain a complete antigen; diluting the complete antigen with normal saline, mixing the diluted complete antigen with a complete Freund's adjuvant, carrying out an emulsion treatment so as to obtain primary immunized antigen emulsion, subjecting a rabbit to a primary immunization by using the primary immunized antigen emulsion, then subjecting the rabbit to a strengthened immunization, wherein the strengthened immunization uses a strengthened immunized antigen emulsion, which is prepared by the following steps: mixing the diluted complete antigen with an incomplete Freund's adjuvant, and then carrying out an emulsion treatment so as to obtain the target product; collecting the blood of the immunized rabbit, when the antiserum titer of rabbit arrives at 1 / 10000; making the blood blocks fully contract to completely separate out the antiserum, then collecting the antiserum, and subjecting the antiserum to a centrifugation treatment so as to obtain a supernate. The antibody prepared by the invention has a strong specificity, and can be used for detection and analysis of silk fibroin in textile, and the like.

Description

technical field [0001] The invention relates to a technology for preparing polyclonal antibodies, in particular to a technology for preparing specific antibodies to silkworm silk protein. Background technique [0002] The silkworm is an important economic insect in my country, and its purpose is to produce silk. Silk is mainly composed of two proteins, silk fibroin and sericin, but sericin protein falls off during the silk reeling process. Therefore, the main component of silk fabrics and silk products is silk fibroin. On the one hand, there are many counterfeit silk products in the textile market at present, which violates the rights of consumers and seriously affects the long-term good reputation of Chinese silk. Therefore, it is necessary to establish scientific and rigorous detection and analysis methods. On the other hand, in the field of research on the origin of silk, there is an urgent need for more scientific analysis methods, because silk fabrics buried in tombs o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06
Inventor 郑秦沈运旺吴小锋郑海玲周旸
Owner ZHEJIANG UNIV
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