Isolation And Identification Of IBRV And Prokaryotic Expression Its Glycoprotein D

Infectious bovine rhinotracheitis(IBR),which was caused by the Bovine herpersvirus-1(BHV-1),was acute, pyrexic and contagious, caused severe respiratory and genital infections in cattle and incurred great economic loss to the global cattle husbandry. IBR was listed as the B disease by OIE, and was the main object quarantined of the animals imported to China.BHV 1 genome encodes eleven glycoproteins. among them, glycoprotein B and glycoprotein D were the most highly conserved herpesvirus glycoprotein, have been shown to be essential for are involved in crucial steps during virus infection, in particular attachment to and penetration into target cells and direct cell-to-cell spread. They can induce high titer cellular and humoral immune respone, especially gD, it induces a more consistent and stronger cellular immune response than gB. And gD triggers apoptosis in peripheral blood mononuclear cells and bovine lymphoma cells, which could contribute to immunodepression in infected animals. Based on them, threes aspects of research works have been carried out in this paper.1 Isolation and identification of infectious bovine rhinotracheitis virusA infectious bovine rhinotracheitis(IBR) virus was isolated from a sick cow nasal swabs with (MDCK) cells, and the neutralizing antibody against the isolated virus were detected by the standard IBRV specific serum. A specific pair of primers were designed according to the gene sequence of IBRV gB gene publicated in GeneBank. The gB gene of the IBRV strain was amplified by PCR method, and the sequences was analyzed.The results showed that obvious characteristics of the cytopathic effect(CPE) generated in MDCK, such as grape-like clusters of rounded cells gathered around a hole in the monolayer; sometimes giant cells with several nuclei may be observed;The virus can be neutralized completely by the standard IBRV specific serum. Specific band can be amplicified by PCR with IBRV gB specific primers and the sequencing result showed that the sequence of the isolated virus was coincided with the published IBRV gB sequences. The results indicated that the isolate was IBRV, and be named IBRV XA strain.2 Establishment and Application of PCR for Detection Methods of IBRVA specific pair of primers were designed according to the gene sequence of infectious bovine rhinotracheitis virus (IBRV)gB gene publicated in GeneBank. The gB gene of the IBRV strain was amplified by PCR method, and the motive fragment of amplification is 1182bp. The PCR detective method was verified by specificity and sensibility tests. Meanwhile, Using the method to detect the serum samples which come from clinic bovine and part of manufacturers in China. The results showed that the method not only has high specificity, but also can examine virus with 103.4TCID50/0.1mL , The minimum detectable amount of virus DNA was 2.16ng/20ul。There were 12 positive samples in 98 sera, positive rate was 12.2%,among them clinic bovine and part of manufacturers in China were 12.8% and 8.3% Respectively. And it was considered to be the foundation for detecting and epidemiologic survey of IBRV.3 Prokaryotic expression of glycoprotein D of IBRVA specific pair of primers were designed according to the gene sequence of infectious bovine rhinotracheitis virus (IBRV)gB gene publicated in GeneBank. gD gene was amplified by PCR and cloned directly into the pET-32a(+),the recombinant prokaryotic expression plasmid pET-32a-gD was successfully constructed. pET-32a-gD was transformed into E.coli BL21(DE3), and induced with IPTG, the expressed products(gD proteins) had antigenicity same as IBRV which were analyzed with SDS-PAGE and Western blotting.