Isolation And Identification Of Infectious Bovine Rhinotracheitis Virus And Prokaryotic Expression Of Partial Glycoprotein B

Infectious bovine rhinotracheitis （IBR） is an acute, thermal property and contagious infectious disease of cattle, caused by the bovine herpes virus type â…  （BHV-1）.The disease spreads throughout the world, it damages the respiratory and reproductive system of cow mainly, which causes enormous losses to cattle industry,and its latent infection maks the prevention and control of the disease harder and difficult to eradicate. Glycoprotein B with highly conservatism encoded by BHV-1 plays an important role in virus adsorption, the invasion process and the infected cells, and it is an essential structural protein for the replication of virus.The protein can induce humoral and cellular immune response, and it is one of the preferred specific antigen for detecting IBRV. Based on this, three aspects following were carried in this article:1 Serological and etiological investigation of infectious bovine rhinotracheitisTo explore the prevalence of IBR in dairy herd in Ningxia region,470 cow serum and 16 suspected clinical samples infected by IBR from 18 large-scale dairy farms in Ningxia region were collected in this study. Enzyme-linked immunosorbent assay and PCR technology were used to detect BHV-1 serology and molecular biology respectively.The result showed that 338 kind of all serums were BHV-1 antibody positive serum, the average positive rate was 71.9%; And 7 kind of clinical samples were BHV-1 positive.lt indicated that infectious bovine rhinotracheitis was popular in Ningxia region and this study provided a scientific basis for epidemiological study and control of IBR in Ningxia region.2 Isolation and identification of infectious bovine rhinotracheitis virusAfter freezing and thawing the BHV-1 positive samples for a few times, inoculate the supernatant to MDBK cells to separation and culture the virus. It showed that a strain of virus with an obvious cell lesions was isolated from a calf organs. It confirmed that the infectious bovine rhinotracheitis virus of cow was successfully separated in Ningxia region by virus neutralization test, polymerase chain reaction （PCR） and nucleic acid sequencing,and was named IBRV YF₁5 strains.3 Prokaryotic expression of partial glycoprotein B of IBRVThe antigenicity of glycoprotein B was analysised by using biology software DNAStar, select a effective fragment in extracellular area that can fully induced protective immune responses. The specific primers was designed and synthesized contraposing the fragment, the viral IBDV YF₁5 genome extractived was used as template to amplify the purpose fragment gBV. Insert purpose fragment into the expression vector pET32a to constitute the recombinant plasmid,after determining that the amplification was accurate,and then transformed into E.coli BL21 to induced express.Specific bands which was consistent with the expected purpose fragment size was obtained by SDS-PAGE electrophoresis analysis.Recombinant proteins was highly effective expressed under the optimized induced condition, and based on the analysis of Western-blotting,it showed that recombinant protein gBV could react specifically with IBR positive serum but the negative serum.In this study, the gene fragment located in the gB extracellular region has been successfully cloned, and effective specific antigen as to diagnostic IBRV was got based on the gene fragmenta highly expressed in E. coli.it laid a foundation for studying the function of gB protein further and establishing an effective ELISA diagnostic method for infectious bovine rhinotracheitis.