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

A specific antibody and silk technology, applied in the field of polyclonal antibody preparation, to achieve the effect of sensitive, specific, fast identification method and high sensitivity

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

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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 was determined that the "GAGAGSGAGAGS" polypeptide fragment had the highest frequency of occurrence, with a total of 170 places. It was the characteristic polypeptide of silkworm silk fibroin, so the antigen was prepared from this fragment.

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

[0016] Complete Antigen Synthesis:

[0017] Use the Fmoc method to synthesize the hapten (that is, the "CGAGAGSGAGAGS" polypeptide sequence), and add cysteine ​​to the N-terminal of the "CGAGAGSGAGAGS" polypeptide sequence to couple the polypeptide to KLH. Through the action of the cross-linking agent, the sulfhydryl group on the cysteine ​​at the N-terminal of "CGAGAGSGAGAGS" forms a covalent bond with the KLH primary amine, thereby coupling the polypeptide and KLH to obtain...

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Abstract

The invention discloses a method of preparing a bombyx mori silk fibroin specific antibody by utilizing a characteristic polypeptide. The method comprises the following steps: synthesizing a "CGAGAGSGAGAGS" polypeptide sequence by utilizing an Fmoc method, coupling the polypeptide with keyhole limpet hemocyanin (KLH) through the cysteine on the N terminus of the polypeptide 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, adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain a 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 adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain the target product; collecting the blood of the immunized rabbit, when the antibody titer in the rabbit blood sample reaches 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.

Description

technical field [0001] The invention relates to a method for preparing a polyclonal antibody, in particular to a method for preparing a silk fibroin specific antibody of silkworm. 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 or ruins ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06
Inventor 郑秦吴小锋郑海玲周旸
Owner ZHEJIANG UNIV
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