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

A technology of thyrotropin and expression products, applied in the field of genetic engineering, can solve the problems of only measuring TRAb, not being suitable for routine use, and not being able to be promoted, and achieve the effect of sensitive method

Inactive Publication Date: 2007-04-25
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

[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. Biolo

Method used

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

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Experimental program
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Embodiment Construction

[0057] 1. Cloning of human thyrotropin receptor extracellular region amino terminal, middle segment and carboxy terminal gene, construction and identification of expression plasmid

[0058] (1) Cloning of amino-terminal, middle segment and carboxyl-terminal genes of extracellular region of human thyrotropin receptor

[0059] The first strand of human thyroid cDNA was synthesized by reverse transcription of total RNA extracted from the excised thyroid tissue of patients with Graves' disease by TRIzol one-step method, and the hTSHR-N terminal and hTSH-C were amplified by RT-PCR with self-designed primers end and hTSHR-M encoding gene. Taking the nucleic acid molecular weight standard as a reference, identify the PCR amplification product by 1.5% agarose gel electrophoresis, and the results show that a clear and specific electrophoresis band can be seen at 462bp, 417bp and 405bp respectively, which is consistent with the expected gene fragment size (see figure 1). In Figure 1: ...

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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-N terminal, 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...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/09G01N33/68
Inventor 方佩华陈慧李宁王少艳谭建
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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