Gene expression product of extro-cellular domain amino end of human thyrotropin receptor, its preparing method and application in enzyme immune technology

Abstract

The invention discloses the expressing product of human thyrotropin receptor extracellular domain amino terminus gene and its preparation methods and the application of the product in the enzyme immunoassay technology. The purpose of this invention is to divide TSHR-ecd cDNA into three regions(hTSHR-N terminal, hTSHR-C terminal and hTSHR-M terminal), clone them respectively, especially the hTSHR-Nterminal, construct expression vector, express in E.coli, and establish TRAb (including TSAb. TSBAb) ELISA technology taking the protein of this gene engineering fragmentas antigen. The said invention develops novel channel for further research of TSHR antigen determinant and settles foundation for the ultimate transformation into TSAb, TSBAb clinical diagnostic kit.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an amino-terminal gene expression product in the amino-terminal, carboxyl-terminal and middle segment of the extracellular region of human thyrotropin receptor, a preparation method thereof and the enzyme immunoassay and luminescent enzyme immunoassay technology of the product in the application. Background technique [0002] Autoimmune thyroid disease (AITD) is a kind of frequently-occurring disease, including Graves' disease (GD), chronic lymphocytic thyroiditis (HT), etc., and its pathogenesis has not yet been fully understood. [0003] Thyroid-stimulating hormone receptor (TSHR) is a glycoprotein present on the membrane of thyroid follicular epithelial cells (TEC). After it binds to thyroid-stimulating hormone (TSH), it regulates the growth, differentiation, and synthesis and release of thyroid hormones of TEC. function. On the basis of environmental and genetic f...

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Problems solved by technology

[0004] Current studies suggest that TSAb is mainly bound to the amino-terminal (N-terminal) of TSHR-ecd, and it has been reported that aa22-61 at the N-terminal of TSHR-ecd is an important target region of TSAb; the target site of TSBAb is concentrated in the TSHR-ecd Carboxyl terminal (C-terminal), in which Cys 301, Cys381 and Try 390 are the key sites for TSBAb binding; reports on the epitope of the TSHR-middle segment are very limited. The current research on the epitope of TSHRAb mainly comes from gene deletion or replacement , the binding reaction between synthetic peptides and antiserum from AITD patients and animal models, and the research results of genetically engineered expression products, but the former two lacked or could not correctly reflect the spatial conformation of hTSHR-ecd, and obtained hTSHR protein as hTSHR protein epitope research material has become a bottleneck for in-depth research in this field. Because the distribution of TSHR on the thyroid follicular epithelial cell membrane is very rare and extremely unstable, it is difficult to directly obtain a sufficient amount of TSHR from TEC for related research ; Although TSHR has been successfully expressed in the eukaryotic system, and the expression product of this system overcomes the problem of TSHR protein spatial conformation, its expression yield still cannot meet the requirements of TSHR epitope due to the shortcomings of low yield, relatively complicated process, and high production cost. Relatively speaking, the prokaryotic expression system has mature technology, high yield, simple process, and low production cost. It is still the best way to obtain a large amount of target protein in this research. The soluble hTSHR full-length or extracellular domain protein has been expressed in Escherichia coli. Our laboratory has reported that the soluble TSHR-ecd protein has been successfully expressed in E. coli, and the yield of the expressed product has gradually increased after repeated practice. The epitopes of the two autoantibodies are still difficult to distinguish their exact sites. After the gene is roughly separated and expressed, the results obtained by the study of the antigenic determinants will be better than those obtained by the synthetic peptide method. The results are more convincing. Japanese scholar Akira Nakai and others have divided the TSHR extracellular region into two segments for prokaryotic expression, but so far, there is no expression of the hTSHR extracellular region in three segments (amino-terminal, carboxy-terminal and middle segments) report

[0005] At present, there are two commonly used TRAb detection techniques in foreign countries: 1. Radioreceptor analysis, its disadvantage is that it can only measure TRAb and cannot distinguish TSAb from TSBAb

2. Biological analysis: TSAb and TSBAb can be distinguished, but the operation is complicated and not suitable for routine use

And its commodity medicine box is all very expensive, though domestic introduction, can't popularize

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