The use of the highly active expression duck Tembusu virus e protein core antigenic domain protein

A duck Tembusu virus, core antigen technology, applied in the field of protein analysis, preparation and diagnosis, can solve the problems of low sensitivity and accuracy, and achieve the effect of strong control, high sensitivity and high accuracy

Active Publication Date: 2015-12-09
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the sensitivity and the problem that the accuracy is not high in the ELISA detection method of Tembusu virus in the above prior art, the present invention provides a kind of highly active Application of the expressed Duck Tembusu virus E protein core antigenic domain protein in the ELISA detection method of Tembusu virus

Method used

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  • The use of the highly active expression duck Tembusu virus e protein core antigenic domain protein
  • The use of the highly active expression duck Tembusu virus e protein core antigenic domain protein
  • The use of the highly active expression duck Tembusu virus e protein core antigenic domain protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The acquisition of embodiment 1 antigenic protein

[0030]The expression of the core antigen domain of Duck Tembusu virus E protein (hereinafter referred to as EDⅢ) is based on the determination of the core antigen domain, and the PCR amplification expression primer EDⅢexp-1:5' of duck Tembusu virus E protein DⅢ was firstly designed -CATGCCATGGAAAGGCATGACCTACCCGATGTG-3' (contains NcoI restriction site), EDⅢexp-2:5'-CCGCTCGAGACTTCTATGCCACTGGTACCT-3' (contains XhoI restriction site), used for cloning duck Tembusu virus EDⅢ. After the PCR product was digested with NcoI and XhoI, the PCR product was ligated into pET32a, which was also digested with the same double enzyme, to construct the recombinant expression plasmid pET32a / EDIII, and then transformed into Escherichia coli expression strain RosettatagamiB (DE3) for highly active expression of EDIII. SDS-PAGE results showed that when the temperature was 18°C ​​and the IPTG concentration was 0.2mM overnight induction cult...

Embodiment 2

[0033] The optimal coating concentration of duck Tembusu virus EDⅢ indirect ELISA method, the optimal serum dilution, the optimal coating conditions of antigen, the working concentration and time of the enzyme-labeled secondary antibody were obtained by the following methods:

[0034] (1) Determination of optimal antigen coating concentration and optimal serum dilution

[0035] The concentration of antigen and serum was determined by square array titration. Use 20mM Tris (pH 7.4) to serially dilute the EDⅢ antigen protein, the final concentrations are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 100 μL per well horizontally coated microtiter plate, 4 ℃ overnight. Then use PBST solution to wash three times, add blocking solution, and block at 37°C for 2h. Negative and positive sera were diluted longitudinally on the microtiter plate starting from 1:2000. The dilutions were 1:2000, 1:4000, 1:8000, and 1:16000. Plate 3 times. Then add 2000-fold diluted goat anti-duck enzyme-l...

Embodiment 3

[0046] Example 3 Sensitivity of duck Tembusu virus EDⅢ indirect ELISA

[0047] Choose a 96-well ELISA plate, add 100uL 0.5ug / ml EDⅢ protein to each well, and use it to coat the ELISA plate. After 30min at room temperature, overnight at 4°C, wash with PBST three times, add blocking solution, and block at 37°C. 2h. Add 1:2000-1:32000 times diluted DTMUV positive serum, 100uL per well, react at room temperature for 0.5h, wash the plate 3 times with PBST, then add 100uL1:5000 diluted rabbit anti-duck enzyme-labeled secondary antibody, room temperature for 0.5h, PBST Wash the plate 3 times, add 100uL 1mg / mLTMB substrate at the end, add stop solution after 15min at room temperature, measure OD450 value in microplate reader, and use the calculation of OD450 detection value-negative control mean value / positive control mean value-negative control mean value method, when the obtained ratio is greater than or equal to 0.2, it is positive, and when it is less than or equal to 0.2, it i...

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Abstract

The invention relates to a protein analyzing, preparing and diagnosing technology in the technical field of biotechnology and relates to application of efficiently and actively expressed protein in a duck tembusu virus E protein core antigen domain. A base sequence of the duck tembusu virus E protein core antigen domain is shown as a sequence 1 in a sequence table; an indirect ELISA detection method which is high sensitivity, accuracy and controllability is built on the basis of screening DTMUV antigen epitope core area; powerful tools and technical support are provided for effectively preventing and controlling transmission of the DTMUV.

Description

technical field [0001] The invention relates to protein analysis, preparation and diagnosis technology in the field of biotechnology, and relates to the application of highly active and highly active expressed duck Tembusu virus E protein core antigenic domain protein. Background technique [0002] In 2010, a new type of duck-derived flavivirus broke out in large-scale duck farms in my country and spread rapidly across the country, causing huge economic losses to the duck industry in my country. The pathogen - duck Tembusu virus (Duck Tembusuvirus, DTMUV) is a member of the family Flaviviridae and the genus Flavivirus, and is a single-stranded positive-sense RNA virus with an envelope and no segments. Ducks infected with DTMUV mainly suffered from neurological symptoms, reduced feed intake, severe decline in egg production or even extinction, and a large number of deaths. Ducks infected with DTMUV are prone to secondary viral hepatitis, infectious serositis, Escherichia col...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/56983G01N33/68
Inventor 张琳黄庆华张秀美许传田杨少华黄艳艳
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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