Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain

A duck Tembusu virus and core antigen technology is applied in the biological field to achieve the effects of strong manipulation, high sensitivity and strong accuracy

Active Publication Date: 2014-12-10
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

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  • Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain
  • Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain
  • Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain

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Experimental program
Comparison scheme
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Embodiment 1

[0031] The acquisition of embodiment 1 antigenic protein

[0032]The expression of the core antigen domain of Duck Tembusu virus E protein (hereinafter referred to as E DⅢ) was based on the determination of the core antigen domain, and the PCR amplification expression primer E DⅢ exp-1 of Duck Tembusu virus E protein DⅢ was firstly designed : 5'-CATGCCATGGAAAGGCATGACCTACCCGATGTG-3' (including NcoI restriction site), E DⅢ exp-2: 5'-CCGCTCGAGACTTCTATGCCACTGGTACCT-3' (including XhoI restriction site), used for the cloning of Duck Tembusu virus E DⅢ . After the PCR product was double-digested with NcoI and XhoI, it was connected into the same double-digested pET32a to construct the recombinant expression plasmid pET32a / EDIII, and then transformed into the Escherichia coli expression strain Rosetta gami B (DE3) for highly active expression of EDIII. The results of SDS-PAGE showed that when the temperature was 18 ℃ and the concentration of IPTG was 0.2mM, the high-efficiency solu...

Embodiment 2

[0035] Duck Tembusu virus E DⅢ indirect ELISA method The optimal coating concentration of the antigen, the optimal serum dilution, the optimal coating condition of the antigen, the working concentration and time of the enzyme-labeled secondary antibody were obtained by the following methods:

[0036] (1) Determination of the optimal coating concentration and serum dilution of the antigen

[0037] The concentration of antigen and serum was determined by square array titration. Use 20mM Tris (pH7.4) to serially dilute the E DⅢ antigen protein, the final concentrations are 1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml, 100μL per well, horizontally coated with a microtiter plate , overnight at 4°C. 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 microplate plate starting from 1:2000, the dilutions were 1:2000, 1:4000, 1:8000, 1:16000, and 100??L was added to each well, and the ef...

Embodiment 3

[0048] Example 3 Sensitivity of duck Tembusu virus E DⅢ indirect ELISA

[0049] Choose a 96-well ELISA plate, add 100?L 0.5?g / ml EDIII protein to each well, and use it to coat the ELISA plate. The coating solution is reacted at room temperature for 30 minutes and then overnight at 4°C, then washed three times with PBST solution, and added Blocking solution, block at 37°C for 2h. Add 1:2000-1:32000 times diluted DTMUV positive serum, 100?L per well, react at room temperature for 0.5h, wash the plate 3 times with PBST, then add 100?L 1:5000 diluted rabbit anti-duck ELISA II Antibody, at room temperature for 0.5h, wash the plate 3 times with PBST, finally add 100??L1mg / mL TMB substrate, add stop solution after 15min at room temperature, measure the OD450 value in a microplate reader, and judge the result using the OD450 detection value-negative control The calculation method of mean value / positive control mean value-negative control mean value, when the obtained ratio is great...

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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 (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 non-segmented 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 coli and ...

Claims

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Application Information

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Patent Type & Authority Applications(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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