Method for preparing tussah silk fibroin antibody using characteristic hexapeptide

A tussah silk fibroin protein and antibody technology, applied in peptide preparation methods, chemical instruments and methods, from serum immunoglobulin, etc., can solve silk protein degradation, difficult to detect silk fibroin, macromolecular chain breaks, etc. problem, to achieve the effect of short immunization cycle, less antigen dosage and high sensitivity

Active Publication Date: 2019-03-05
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the past, there were many methods of identifying ancient silk fabrics. However, as an organic polymer material, silk has been in the environment of tombs or ruins for a long time, and it will inevitably be affected by various factors, resulting in problems such as degradation of silk proteins and breakage of macromolecular chains. It is difficult to detect the presence of silk fibroin by some traditional methods

Method used

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  • Method for preparing tussah silk fibroin antibody using characteristic hexapeptide
  • Method for preparing tussah silk fibroin antibody using characteristic hexapeptide

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Effect test

Embodiment 1

[0030] A method for preparing tussah silk fibroin antibody using characteristic hexapeptide, which comprises the following steps:

[0031] Complete antigen synthesis:

[0032] The hapten (ie "CGSGAGG" polypeptide sequence) was synthesized by the Fmoc method, and cysteine ​​was added to the N-terminal of the "CGSGAGG" polypeptide sequence to couple the polypeptide with keyhole limpet hemocyanin (KLH). Through the action of a cross-linking agent, the sulfhydryl group on the N-terminal cysteine ​​of "CGSGAGG" forms a covalent bond with the primary amine of KLH, thereby coupling the polypeptide and KLH to obtain a complete antigen.

[0033] The complete antigen was diluted to 2 times the final concentration with normal saline, and the diluted complete antigen was evenly mixed with QuickAntibody-Rabbit 5W adjuvant at a volume ratio of 1:1 to obtain the reagent for primary immunization. The reagents used for the booster immunization were the same as those used for the primary immun...

Embodiment 2

[0042] A method for preparing tussah silk fibroin antibody using characteristic hexapeptide, which comprises the following steps:

[0043] Complete antigen synthesis:

[0044] The hapten (ie "CGSGAGG" polypeptide sequence) was synthesized by the Fmoc method, and cysteine ​​was added to the N-terminal of the "CGSGAGG" polypeptide sequence to couple the polypeptide with keyhole limpet hemocyanin (KLH). Through the action of a cross-linking agent, the sulfhydryl group on the N-terminal cysteine ​​of "CGSGAGG" forms a covalent bond with the primary amine of KLH, thereby coupling the polypeptide and KLH to obtain a complete antigen.

[0045] The complete antigen was diluted to 2 times the final concentration with normal saline, and the diluted complete antigen was evenly mixed with QuickAntibody-Rabbit 5W adjuvant at a volume ratio of 1:1 to obtain the reagent for primary immunization. The reagents used for the booster immunization were the same as those used for the primary immun...

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Abstract

The present invention discloses a method for using a characteristic hexapeptide for preparing a tussah fibroin protein antibody, a synthetic sequence is a ''GSGAGG'' hexapeptide, the hexapeptide is coupled with keyhole limpet hemocyanin to obtain a full antigen; the full antigen is diluted with normal saline, the diluted full antigen is mixed evenly with Quick Antibody-Rabbit 5 w adjuvant, primary immunization is performed on a rabbit, then booster immunization is performed on the rabbit, reagents used for the booster immunization and the primary immunization are same; when titer of the antibody in a blood sample of the rabbit reaches 1: 10,000, blood of the immunized rabbit is collected, blood clots are fully contracted, antiserum is completely precipitated, then the antiserum is collected antiserum and centrifuged to obtain a supernatant, and the supernatant is sub-packed for standby use. The method is simple and quick, and the prepared antibody is highly specific and can be used to detect silk fibroin in textiles.

Description

technical field [0001] The invention relates to a preparation method of polyclonal antibodies, in particular to a method for preparing tussah silk fibroin antibodies by using characteristic polypeptides. Background technique [0002] Silk is a kind of natural polymer protein fiber, which is mainly divided into two categories: domestic silkworm silk and wild silkworm silk. Wild silkworms are also called tussah silkworms. They get their name because they like to eat oak leaves. They can reel silk from their cocoons and are mainly used for weaving tussah silk. my country is the first country to use tussah silkworms and raise tussah silkworms, but its origin has always been a hot and difficult research point, and a more scientific method is urgently needed to identify silk fabrics in early tombs or remains. In the past, there were many methods of identifying ancient silk fabrics. However, as an organic polymer material, silk has been in the environment of tombs or ruins for a l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/06C07K1/22
Inventor 游秋实王秉彭志勤胡智文
Owner ZHEJIANG SCI-TECH UNIV
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