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Novel duck reovirus recombinant sigma B protein antigen, preparation method and application

A technology of recombinant protein and duck retort, which is applied in the direction of peptide preparation method, botany equipment and method, biochemical equipment and method, etc., can solve the problems of cumbersome operation, unsuitability for veterinary clinical and grass-roots application, and achieve low production cost , wide market application prospects and high detection sensitivity

Inactive Publication Date: 2012-09-19
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

[0004] At present, there is no other detection method except for the serum neutralization test which can be used to detect the new duck reovirus disease antibody. Although the neutralization test is a classic method for antibody detection and has good specificity, it is cumbersome to operate and is not suitable for veterinary clinical and Grassroots application

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  • Novel duck reovirus recombinant sigma B protein antigen, preparation method and application
  • Novel duck reovirus recombinant sigma B protein antigen, preparation method and application
  • Novel duck reovirus recombinant sigma B protein antigen, preparation method and application

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Embodiment Construction

[0030] The present invention will be further described below in conjunction with specific examples. (but not a limitation of the invention).

[0031] 1. Design specific primers for amplifying the S3 gene of the NDRV NP03 isolate:

[0032] NDRV-NP03-S3F (upstream primer): 5'-TGCAAGGATCCATGGAGGTGCGTGTGCC-3'

[0033] NDRV-NP03-S3R (downstream primer): 5'-ACCCAGGTCGACTTACCACCTAC-3'

[0034] 2. NDRV NP03 isolate culture and RNA extraction

[0035] The seed virus of NDRV NP03 isolate was inoculated into 12-day-old non-immune Muscovy duck embryos through the allantoic cavity route, incubated in a 37°C incubator, and the allantoic fluid of Muscovy duck embryos was collected between 48h and 72h, centrifuged and the supernatant was taken for later use. NDR VRRNA was extracted according to the instructions of the Trizol kit; the extracted RNA was used for RT-PCR.

[0036] 3. RT-PCR of S3 gene of NDRV NP03 isolate

[0037] (1) Reverse transcription of NDRV NP03

[0038] Add 7 μL of ...

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Abstract

The invention discloses preparation of novel duck reovirus recombinant sigma B protein antigen, and application of the novel duck reovirus recombinant sigma B protein antigen in detection of a novel duck reovirus antibody. With RNA (ribonucleic acid) of the novel duck reovirus (NDRV) as a template, a nucleotide complete sequence of an NDRV S3 gene coding region can be amplified through a reverse-translation polymerase-chain reaction, is directionally subcloned to a fusion expression vector of the prokaryotic expression vector pET-30a-C(+) of escherichia coli, and transformed into the host cell of the Escherichia coli BL21 (DE3), and is subjected to inducible expression with IPTG (isopropyl-beta-d-thiogalactoside), purification of Ni-NTA Agarose, inclusion body refolding to obtain the expressed NDRV alpha B recombinant protein antigen. The expression mode of the protein is fusion protein (His-alpha B) with molecular weight being about 44kU; and immunoblotting assays prove that the protein has immunoreactivity similar to natural protein..

Description

technical field [0001] The invention relates to the preparation of recombinant σB protein antigen of novel duck reovirus and its application in detecting antibody of novel duck reovirus disease. technical background [0002] Duck hemorrhagic necrotizing hepatitis is a kind of duck reovirus (Novel duck reovirus, NDRV) caused by the new type of duck reovirus (NDRV) in the family Reoviridae. It is characterized by irregular liver necrosis and hemorrhage. The main characteristic of the blight. Different breeds of ducks are susceptible, and the younger the age or the higher the morbidity and mortality when they are concurrently infected, the majority are 5 to 10 days old, the course of disease is 5 to 7 days, the incidence rate is 5% to 20%, and the mortality rate is 2% to 15 days. %. The pain is widely prevalent in waterfowl breeding areas in southern my country, and the infected areas have a tendency to expand year by year, causing certain economic losses to the waterfowl bre...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C12N15/46C12N15/70G01N33/569
Inventor 王劭陈少莺林锋强陈仕龙朱小丽程晓霞李兆龙
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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