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Isolation and Identification of Goose Reovirus and Cloning、Expression of Its SigmaC and Preparation of Polyclonal Antibodies

Author: WangChen
Tutor: XuJianSheng
School: Yangzhou University
Course: Preventive Veterinary Medicine
Keywords: GRV σC gene clone and expression polyclonal antibody
CLC: S852.65
Type: Master's thesis
Year: 2013
Downloads: 19
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Abstract


A goose farms in Jiangsu happened a new infectious disease which infects goslings only.The pathological change of the disease is characterized by hemorrhagic necrotizing hepatitis. We inoculate SPF chicken embryos with the treated collections in advance which come from diseased goslings’s necrosic liver, and identify virus through the way of clinical symptoms, autopsy lesions and sequence comparison. The results of experiments show that the separation of this virus can cause regularly death and characteristic pathological changes of10day-old SPF chicken embryos,14day-old SPF goose embryos,which is inoculate by the way of chorioallantoic membrane.But the chicken embryos and goose embryos do not die which is inoculate by the way of allantoic cavity.The allantoic fluid from dead chicken embryos is inoculated to chicken fibroblast cells, but we haven’t seen any obvious pathological changes in cells. Cells are growing well. We inoculate allantoic fluid,chorioallantoic membrane,embryo body with virus to chicken embryos and goose embryos to determine median lethal dose (LD50),and the result are10-27/0.2mL、10-29/0.2mL、10-3.7/0.2mL、10-4.0/0.2mL、10-3.4/0.2mL、10-3.8/0.2mL,respectively.Inoculate1,30,80day-old geese with allantoic fluid from dead chicken embryos,1day-day goslings dead partly, others are good with no obvious clinical symptoms. In addition, this virus could not agglutinate gallinaceous akaryocyte, so it had no compendency.Sigma C protein of ARV is encoded by the third open reading frame of S1gene, existed in various forms, and it is the main part of the capsid protein. Sigma C protein also can induce neutralizing antibody production. Sigma C protein plays a main role on the pathogenicity of the virus, Missing part of the third ORF genes can lead the decline of virus adsorption.According to the published sigma C gene on the NCBI, design a pair of specific primers of gene sequence to detect goose reovirus. Partial sigma C gene of GRV was amplified by adopting the method of RT-PCR. Connected the sigma C gene to T carrier, transform the product into e.coli DH5a competent cells, choose white colonies in the A+LB agar plate with IPTG and X-gal, extract plasmid, Preliminary judge by agarose gel electrophoresis, and send gene to be sequenced, the cloning is867bp. Use BamH I and Sal I cleavage sigma C gene from T vector plasmid, recover sigma C gene fragments, recombinated the gene into prokaryotic expression vector pGEX-6p-1. The recombinant plasmids were transformed into E.coli BL21(DE3)and then induced by0.01mM IPTG last for5hours in37℃.The result of SDS-PAGE analysis showed that σC protein was successfully expressed, and its molecular weights was55KD, named GST-σC. The immunity reactivity of the recombinant proteins was confirmed by Western-blot method.Two New Zealand rabbits were immunized with purified protein for producing polyclonal antibody. After three times immunization, detect antibody titer by indirect Elisa. High titer of antibody is the first premise to validate expression and function, because the disease was found not long, the main methods of diagnose GRV infections are virus separation and AGP. The expression of recombination protein and preparation of polyclonal antibody may facilitate us to study the immunologic functions of the protein and monoclone antibody and it provided a useful tool for epidemiological investigation of GRV.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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