Estrogen receptor α gene, encoded protein and enzyme-linked immunoassay method in rice field eel

Abstract

The invention discloses a monopterus albus estrogen receptor alpha gene, an encoding protein and an enzyme-linked immunosorbent assay method. The enzyme-linked immunosorbent assay method comprises the following steps: obtaining an ER alpha sequence in monopterus albus gonad by adopting an RT-PCR method, selecting cDNA sequence corresponding to an amino acid sequence with stronger antigenicity as a template to clone the sequence of which length is 945bp, cloning the sequence into a pMAL-c2x expression vector, obtaining purer recombinant protein with immunocompetence through IPTG induction and Amylose-sepharose columella purification, and obtaining ER alpha expression protein capable of being used as immunogen by adopting FactorXa protein enzyme digestion after further purification. A mice can obtain antiserums with higher titer after being immunized, and an immunohistochemical research is preliminarily conducted, shows that the antiserums can be peculiarly bond with ER alpha protein in the monopterus albus gonad, shows positive reaction on oocyte membrane, and lays a foundation for further researching functions of estrogen, ER alpha of the estrogen and the like in gender development of the monopterus albus. According to the invention, an ELISA assay kit is further prepared by taking the antibody as the basis.

Description

Technical field

[0001] The invention relates to the field of genetic engineering, in particular to the rice field eel estrogen receptor alpha gene, coding protein and enzyme-linked immunoassay detection method. Background technique

[0002] Monopterus albus (Monopterusalbus), also known as eel, mainly lives in Asia. Currently, there is only one species in China, which has high nutritional value. Since the beginning of artificial breeding in the 1970s, wild rice field eel seeds have been captured in large numbers. At the same time, environmental pollution has caused the wild resources of rice field eel to decrease day by day, and there is a serious shortage of cultivated seeds. There is a phenomenon of sexual reversal in the development cycle of rice field eel. When rice field eel begins to develop, it is female. After sexual maturity and spawning, male germ cells begin to develop, forming an intersexual stage, and gradually develop into male rice field eel.

[0003] The phenomenon...

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