Construction Of Recombinant BHV-1 Expressing BRSV G Gene And Characterization Of Truncated Glycocoprotein G Of BRSV Expressed In E. Coli

Infectious bovine rhinotracheitis (IBR), caused by bovine hepervirus-1 (BHV-1), is an acute, feverish and contagious infectious disease of cattle, characterized by hyperpyrexia, anhelation, coryza, antritis, and upper respiratory tract inflammation. IBRV can also cause abortion, fetal death, enteronitis, calf encephalitis, and sometimes causes conjunctivitis and ceratitis. While Bovine respiratory syncytial virus (BRSV) belongs to the pneumovirus genus within the family Paramyxoviridae and is a major cause of respiratory disease in young calves.Both of the diseases are epidemic all over the world, and cause significant economic losses every year. There are some drawbacks of the traditional vaccines. Recently, more and more researchers turn to study bovine hepervirus-1 as the live vector-viral vaccine based on some characteristic of BHV-1. The objective of this study is to construct a recombinant BHV-1 expressing BRSV G gene, providing a platform to develop live vector-viral vaccine expressing exogenous gene. Meanwhile, the truncated G protein of BRSV was expressed in soluble form in E.coli. The purified recombinant protein G1 has good antigencity, and can be used to develop subunit vaccine and diagnosis kits for BRSV.In order to construct the recombinant BHV-1 expressing BRSV G gene, a BHV-1 TK gene deleted transfer vector was constructed with the synthetic G gene coding sequence of BRSV under the control of CMV promoter. The mixtures of parental virus (BHV-1/TK—/LacZ+) DNA and transfer vector was transfected into BT cells using calcium phosphate-mediated transfection. The propagated viruses were harvested after transfection. The recombinant BHV-1 (designated BHV-1/TK—/G+) was obtained by selection for white virus plaques. PCR results showed that G gene was successfully inserted into the genome of BHV-1/TK—/LacZ+. The expression of G gene in infected cells was proved by Western blot and indirect immunofluorescence assay.The G protein was identified as the major attachment protein because antibodies specific to the G protein blocked the binding of virus to cells and it is a major protective antigen of BRSV .Two fragments G1 and G2 of the glycoprotein G gene of bovine respiratory syncytial virus (BRSV) were selected for expression in Escherichia coli based on the analysis of glycoprotein G by DNA Star software. Then the two fragments of glycoprotein G were amplified by PCR with synthesized G gene of BRSV as the template. The amplified fragments G1 and G2 are 570bp and 308bp in length, respectively. The PCR products were cloned into pET30a vector and expressed in soluble form in E. coli after induction of cultured E. coli with IPTG. Both of the recombinant proteins G1 and G2 were purified by immobilized Ni ion affinity chromatography under native conditions. Then the purified proteins were analysed by western blot. The results showed that the purified recombinant protein G1 retained good antigenicity and specificity. But the purified recombinant protein G2 didn't possess biological activity. Antibodies against BRSV were detected in suspected bovine sera by using indirect ELISA and western blot with the purified recombinant protein G1 in China. The purified recombinant protein G1 might be used as antigen for establishing serological methods for diagnosis of BRSV infection. And the purified recombinant protein G1 might also be used for preparing polyclonal and monoclonal antibodies for research on biological functions of glycoprotein G of BRSV.