nAChR-alpha1-ECD protein and preparation method thereof

Abstract

The invention provides a nAChR-alpha1-ECD protein which is characterized in that the amino acid series is SEQ ID 1, wherein the molecular weight of the protein is 28.7kDa and the isoelectric point is 6.0. Moreover, the invention also provides a preparation method of the protein. The method comprises the following steps: S1, performing in vitro synthesis of a cDNA sequence SEQ ID2 corresponding to a SEQ ID 1 amino acid sequence; S2, inserting the SEQ ID 2 sequence into an expression vector pCold II, wherein the insertion site is Nde I / Hind III; S3, converting pCold II-AChR-alpha1-ECD plasmids obtained in the S2 into BL21(DE3) competent cells, performing overnight culture with a panel with penbritin, selecting a monoclonal colony and performing an expression lab-scale test by inductive amplification of isopropyl-beta-D-thiopyran galactoside (IPTG) to obtain an escherichia coli strain expressed by AChR-alpha1-ECD BL21(DE3); S4, performing amplified fermentation on the escherichia coli strain expressed by AChR-alpha1-ECD BL21(DE3); and S5, splitting the Escherichia coli thalli to obtain an inclusion body solution, and performing follow-up separating, purifying and renaturating steps.

Description

technical field [0001] The invention relates to an nAChR-α1-ECD protein and a preparation method thereof, belonging to the field of medical detection. Background technique [0002] The Department of Neurology of Shanghai Changhai Hospital began to carry out clinical trial research on myasthenia gravis (MG) in 1978. It used electric rays and human muscle to extract nAChR antigen and detected myasthenia gravis nAChR antibody (nAChR-Ab). This method had to be discontinued due to the increasingly limited materials. [0003] However, there is currently no human AChR-Ab clinical detection kit for sale on the market, and the old method continues to use animal or human muscle extraction. The operation is complicated and costly, and it cannot meet the needs of a large number of patients in reality. However, gene expression after artificial synthesis directly based on the amino acid sequence of human nAChR, although the target protein can be obtained, is often difficult to separate o...

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Problems solved by technology

[0003] However, there is currently no human AChR-Ab clinical detection kit for sale on the market, and the old method continues to use animal or human muscle extraction, which is complex and costly, and cannot meet the needs of a large number of patients in reality.

However, directly according to the amino acid sequence of human nAChR for gene expression, although the target protein can be obtained, it is often difficult to separate or the purity of the separation is not high and the yield is low due to the nature of the target protein.

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