Grass carp ATG12 polyclonal antibody and preparation method thereof

An ATG12, polyclonal antibody technology, applied in the field of genetic engineering, can solve problems such as unpublished, reported, and no fish ATG12 polyclonal antibody research.

Active Publication Date: 2019-07-26
JIANGXI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, ATG12 gene has been reported in human, mouse, hydra and amoeba, but there is no public report on...

Method used

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  • Grass carp ATG12 polyclonal antibody and preparation method thereof
  • Grass carp ATG12 polyclonal antibody and preparation method thereof
  • Grass carp ATG12 polyclonal antibody and preparation method thereof

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Embodiment Construction

[0042] In order to make it easy to understand the technical means, creative features, objectives and effects achieved by the present invention, the present invention will be further explained below in conjunction with specific drawings.

[0043] The preparation method of grass carp ATG12 polyclonal antibody, the specific steps are as follows:

[0044] (1) Analyze the clone and sequence of grass carp ATG12 gene

[0045] 1) Extract total RNA from grass carp liver

[0046] (1) Quickly take out the frozen grass carp liver from liquid nitrogen and grind it with liquid nitrogen. After the grinding is complete, add 3ml TRIzol Reagent. When it is about to melt, use a 1.5ml EP tube to inhale, 1ml per tube, and place on ice. on;

[0047] (2) Add 20% chloroform (ie 1ml TRIzol Reagent to 0.2ml chloroform) to each tube, shake vigorously for 15s, and stand on ice for 3 minutes to see the layering phenomenon;

[0048] (3) Centrifuge at 12000g for 15min at a temperature of 4°C. The liquid in the EP tub...

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Abstract

A preparation method of a grass carp ATG12 polyclonal antibody comprises the steps: firstly, taking grass carp liver RNA as a template, carrying out reverse transcription to synthesize cDNA, then taking the cDNA as a template, amplifying by PCR with primers and recovering a target fragment, and then constructing an ATG12/pGEX-4T-1 prokaryotic expression vector; transferring the ATG12/pGEX-4T-1 prokaryotic expression vector into competent cells of escherichia coli DH5[alpha], treating, then carrying out ultrasonic crushing and centrifugation to obtain a supernatant and a precipitate, and then purifying the precipitate to obtain purified ATG12 fusion protein; and finally, fully mixing and emulsifying the purified ATG12 fusion protein as an antigen with a Freund's complete adjuvant with the same volume with the ratio of 1:1, immunizing pure New Zealand white rabbits, extracting heart blood, and separating serum, to obtain the ATG12 polyclonal antibody. The antibody has the advantages of high specificity, low cost and short period, and lays the foundation for obtaining the commercially available ATG12 antibody.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a grass carp ATG12 polyclonal antibody and a preparation method thereof. Background technique [0002] Autophagy is a lysosomal-dependent cellular metabolism pathway that is ubiquitous in most eukaryotic cells. It regulates the metabolism of intracellular substances and maintains the stability of the internal environment by degrading intracellular ubiquitin proteins or damaged organelles. It plays an important role in growth, differentiation, aging, and apoptosis (Maiuri et al., 2007). The formation of autophagosomes mainly involves two conservative ubiquitination binding systems, which are necessary for the formation of autophagosomes. One of the ubiquitination binding systems involves the ATG12 gene, which belongs to the family of ubiquitin-like proteins. One of the members, can mediate the expansion of autophagosome membrane, which is essential for the formatio...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/70C12N15/62C07K16/06
CPCC07K16/18C07K14/47C12N15/70C07K2317/10C07K2319/95
Inventor 隗黎丽何丽钟其旺梁惜梅阮记明
Owner JIANGXI AGRICULTURAL UNIVERSITY
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