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A kind of preparation method of specific stimulation antigen

A sexually stimulating and specific technology, applied in the field of genetic engineering, to achieve the effect of good activity and strong specificity

Active Publication Date: 2021-03-12
国家卫生健康委科学技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to overcome the deficiencies in the prior art, the present invention aims to solve the problem of the special antigen preparation method required for preparing antibodies with specific reactions

Method used

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  • A kind of preparation method of specific stimulation antigen
  • A kind of preparation method of specific stimulation antigen
  • A kind of preparation method of specific stimulation antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Strategies using cross-species proteins as specific antigens.

[0030] Taking advantage of the sequence similarity between the subunits of the inhibin B protein molecule and the highly conserved subunit sequences, and the strong cross-species reactivity of antibodies against TGF-β superfamily proteins, cross-species proteins can be used as specificity antigen. Furthermore, zebrafish protein can be used as the specific antigen for preparing human antibodies. Further, zebrafish inhibin B protein can be used as the specific antigen for preparing human inhibin B antibody. Further, the β chain of zebrafish inhibin B protein can be used as the specific antigen for preparing human inhibin B antibody. Furthermore, the zebrafish inhibin B protein β chain can be used as the specific antigen for preparing the human inhibin B β chain antibody.

Embodiment 2

[0031] Example 2 Preparation of zebrafish inhibin B protein.

[0032] S1 optimizes the whole gene sequence of zebrafish inhibin B to obtain the target gene, as shown in SEQ NO.1;

[0033] S2 designed full-length splicing primers, inserted the target gene synthesized in S1 into the vector plasmid pET30a through the restriction sites NdeI and Hind III, and transformed into BL21 (DE3) host cells for expression;

[0034] S3 cultivating the host cells transformed in S3 to obtain a fermentation broth containing a large amount of recombinant protein;

[0035] S4 Separating and purifying the fermentation broth in step S4 to obtain high-purity recombinant inhibin B protein.

Embodiment 3

[0036] Inhibin B protein detection after the purification of embodiment 3

[0037] a. Stability test of inhibin B protein (freeze-thaw test): take a subpackaged inhibin B protein frozen at -80°C, place it in an ice-water mixture until it melts slowly, and there is no abnormal phenomenon after melting, indicating Inhibin B protein freeze-thaw experiments were normal.

[0038] b. Determination of inhibin B protein concentration: Bradford protein concentration determination kit was used to determine the protein concentration.

[0039] c. WB detection of inhibin B protein: For the operation procedure of WB experiment, refer to "Protein Electrophoresis Experimental Technology" written by Guo Yaojun. Test results such as image 3 . Depend on image 3 It can be seen that in the SDS-PAGE electrophoresis pattern of the recombinant inhibin B protein, a bright band appears at 50kDa, and a target band appears at the same position detected by Western-Blot. The purity of the protein as...

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Abstract

The present invention relates to a method for preparing a specific stimulating antigen, using a cross-species protein as the specific stimulating antigen, specifically, utilizing the characteristics of TGF-β antibody cross-species reaction, using zebrafish protein as the specific stimulating antigen for preparing human antibodies. The method for preparing the cross-species protein is as follows: optimizing the whole gene sequence of zebrafish inhibin B; designing full-length splicing primers; constructing recombinant plasmids; transfecting host cells to prepare inhibin B protein, as a preparation of human inhibin B specific antibody antigen. The present invention adopts the method of using a cross-species protein as a specific stimulating antigen, and uses the artificially synthesized cross-species inhibin B protein as an antigen to carry out an immunoassay to obtain a high-purity inhibin B antibody, which provides a method for obtaining human-specific antibodies. A new immunization strategy.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a specific stimulating antigen. Background technique [0002] Inhibin is one of the members of the transforming growth factor β (TGF-β) superfamily and is a dimeric glycoprotein hormone composed of α and β subunits. The relative molecular mass (Mr) of the α subunit is 20000; the relative molecular mass (Mr) of the β subunit is 5000-15000, and there are two subtypes of βA and βB. α and β are joined by disulfide bonds to form inhibin A (αβA) and inhibin B (αβB), and the other two β subunits also form activin A (βAβA), activin B (βBβB) and activin AB (βAβB). INH-B exists in a variety of molecular forms in vivo, including mature (Mr: 32000) and immature (also known as precursor) αβ dimer molecules and free α subunits. The enzyme-linked immunosorbent assay (ELISA) can also detect the alpha subunit preprotein (Pro-αC) in plasma, which may be a seri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/16C07K16/26
CPCC07K14/575C07K16/26C07K2317/24C12N15/70C12N2800/22
Inventor 辛玲于和鸣赵海豹侯丽
Owner 国家卫生健康委科学技术研究所
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