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

An enzyme-linked immunoassay, estrogen receptor technology, applied in the field of genetic engineering

Inactive Publication Date: 2016-06-15
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, enzyme-linked immunoassays require more hormone standards, and conventional pituitary extraction techniques require a large amount of pituitary

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Cloning of eel estrogen receptor α sequence and protein expression

[0035] 1. Material

[0036] 1.1 Experimental materials:

[0037] Experimental fish: Monopterus albus was purchased from Wuxi Zhongqiao Vegetable Market, organized as gonads. Mice: ICR mice purchased from the Experimental Animal Center of Zhejiang University.

[0038] 1.2 Reagents: T4DNA ligase, TaqDNA polymerase, gel recovery kit, EcoRI, HindⅢ, etc. were purchased from Dalian Bao Biological Company, polyacrylamide, RNase inhibitors, etc. were purchased from Shanghai Shenneng Bio-engineering Company, Amylose-sepharose column , FactorXa, and anti-maltose binding protein (MBP) monoclonal antibodies were purchased from New England Biotechnology (Beijing) Company, and other biochemical reagents were all domestically pure. Freund's complete and incomplete adjuvants, ELISA reaction plates, etc. were purchased from Shanghai Shenggong Biological Engineering Co., Ltd.

[0039] 1.3 Instrument: The PCR instrume...

Embodiment 2E

[0063] Example 2 Preparation of ERα receptor protein detection kit

[0064] 2.1 Preparation of expression product antiserum

[0065] After mixing the recovered ERα recombinant protein with an appropriate amount of 1×PBS, take 0.2ml (about 20μg of antigen) and inject 6 female BALB / c mice (4-5 weeks old) into the abdominal cavity. Collect them before immunizing the mice. Part of the blood sample is used as negative serum. The immunization is carried out every two weeks, and blood sampling starts after the first immunization (the mice should be fasted several hours before the blood sampling). The blood samples were collected and placed in a sterile centrifuge tube without anticoagulant and placed in a refrigerator at 4°C overnight to coagulate and precipitate a light yellow antiserum. Transfer the antiserum to another tube, centrifuge the blood clot at 1500g for 10 min, aspirate the supernatant and combine it in the collected antiserum tube. The antiserum part is kept at -70℃ in th...

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/72C12N15/12C12N15/63G01N33/68
CPCC07K14/721G01N33/68G01N2333/723
Inventor 丁炜东邴旭文曹丽萍曹哲明
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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