Acipenser sinensis VTG protein antigen and antibody, and preparation method and application thereof
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A technology of protein antigen and Chinese sturgeon, which is applied in the biological field to achieve the effects of simple cultivation, specific and stable detection reaction, and good immunity
Active Publication Date: 2017-05-10
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Multiple immunizations of Japanese big-eared rabbits resulted in specific antibodies against Chinese sturgeon VTG protein
Method used
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Embodiment 1
[0040] A preparation method of Chinese sturgeon VTG protein antigen and antibody, the steps of which are:
[0041] 1) Extraction and reverse transcription of total RNA from female Chinese sturgeon liver:
[0042] The liver tissue of female Chinese sturgeon preserved in RNAlater was taken out, homogenized in a glass homogenizer, and then total RNA was extracted according to the instructions of the RNA extraction kit. Using the extracted total RNA as a template, reverse transcription was performed according to the instructions of the reverse transcription kit. cDNA. The reaction system is: 42°C for 1hr, heating at 70°C for 10min, and storing at -20°C after the reaction is terminated.
[0043] 2) Amplification and cloning of the target fragment of Chinese sturgeon VTG gene:
[0044] Through the functional search of the VTG amino acid sequence of Chinese sturgeon by BLASTP comparison database, it was found that the 44-612aa region of the amino acid sequence of Chinese sturgeon V...
Embodiment 2
[0054] Specific detection of Chinese sturgeon VTG polyclonal antibody:
[0055] Proteins were extracted from the induced bacteria, the isolated supernatant and the precipitate for western blot detection. The results showed that the polyclonal antibody clearly detected the VTG protein-specific band in the induced bacteria and the isolated precipitate ( figure 2 ), indicating that the prepared Chinese sturgeon VTG polyclonal antibody has good specificity.
Embodiment 3
[0057] A kind of application of Chinese sturgeon VTG protein antigen and antibody in ELISA detection optimal working concentration and dilution ratio, its process is:
[0058] Dilute the recombinant VTG protein with 0.05M carbonate at pH 9.6 to 32.5, 16.25, 8.13, 4.06, 2.03, 1.02, 0.5, 0.25, 0.13, 0.06μg / mL10 concentrations and coat the enzyme plate with 100μl / well , the negative control was not coated with VTG protein, one column was coated with each concentration, and placed at 4°C overnight for coating. After washing 5 times with PBS-T containing 0.1% (v / v) Tween-20, pat dry, add 300 μl of 0.5% (w / v) bovine serum albumin (BSA) blocking solution to each well, and block at 37°C for 1 hour; then Dilute the antiserum at 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 and 1:32000, and add 100 μl / well horizontally to the microtiter plate After reacting at 37°C for 1h, wash 5 times, dilute horseradish peroxidase-labeled goat anti-rabbit IgG 100μl / well with 1:5000, add to th...
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Abstract
The invention discloses Acipenser sinensis VTG protein antigen and antibody, and a preparation method and an application thereof. The preparation method comprises the following steps: 1, preparation of a recombinant Acipenser sinensis VTG protein antigen: 1) designing an upstream primer and a downstream primer, and amplifying a protein domain truncated sequence; 2) subjecting a pMD-18T-VTG plasmid and a Pet32a (+) expression vector to enzyme digestion by using EcoRI and XhoI, then purifying a target fragment, connecting the pMD-18T-VTG plasmid and the Pet32a (+) expression vector into Pet32a (+)-VTG, converting the Pet32a (+)-VTG into DH5a, coating the DH5a onto an ampicillin containing LB culture medium, and carrying out culturing; 3) converting the Pet32a (+)-VTG plasmid into a BL21(DE3) competent cell, coating the BL21(DE3) competent cell onto an ampicillin containing LB solid culture medium, and carrying out culturing; and 4) subjecting collected mycelium precipitate to resuspension precipitation by using an 8M urea containing protein buffer solution, then carrying out filtering, and collecting a supernatant so as to obtain a recombinant protein; 2, obtainment of an Acipenser sinensis VTG protein antibody through immunization of Japanese rabbit with the recombinant Acipenser sinensis VTG protein antigen; and 3, application of the Acipenser sinensis VTG protein antigen and antibody. The Acipenser sinensis VTG protein antibody and the VTG protein antigen provided by the invention have the following advantages: the immunity is good; a titer can reach 1: 1000; detection results have high positive rate; and the clinical applicability is good.
Description
technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a Chinese sturgeon VTG protein antigen and antibody, and also relates to a preparation method of the Chinese sturgeon VTG protein antigen and antibody, and also relates to a use of the Chinese sturgeon VTG protein antigen and antibody. Background technique [0002] Chinese sturgeon (Acipenser sinensis) is a species of sturgeon with long migration distance, fast growth, large body size and largest egg diameter. It has important scientific research value and is listed as a first-class national key protected animal in my country. Due to overfishing, hydraulic construction and other factors, the current population has been greatly reduced, and the current survival problem of Chinese sturgeon species is very serious. The breakthrough of the fully artificial reproduction technology of Chinese sturgeon can get rid of the dependence of artificially multiplied and released see...
Claims
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