Construction Of GC～-/LacZ～+ Gene-deleted Mutant Of Infectious Bovine Rihinotracheitis Virus And Characterization Of Truncated Glycoprotein D Expressed In E.coli

Infectious bovine rhinotracheitis (IBR), caused by infectious bovine rhinotracheitis virus (IBRV), 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. IBR is epidemic all over the world, and causes significant economic losses every year. There are some drawbacks of the traditional vaccines. Recently, more and more researchers turn to study the live vector-viral vaccine based on some characteristic of IBRV. The objective of this study is to construct a recombinant infectious bovine rhinotracheitis virus vector with the gC gene deleted, providing a platform to develop live vector-viral vaccine expressing exogenous gene. Meanwhile, the truncated gD protein was expressed in soluble form in E.coli. The purified recombinant protein tgD has good responsiveness, and can be used to develop subunit vaccine and diagnosis kits for IBRV.Two pairs of primers of gC gene were synthesized based on the genomic DNA sequence of IBRV (GenBank Accession No. NC001847). The upstream and downstream arm sequences of gC gene were amplified by PCR with the two pairs of primers, and were cloned into pMD18-T vector. The recombinant pUgC and pDgC were achieved by identification by digestion and sequencing. The upstream arm, digested from pUgC with XbaⅠand NsiⅠ, was cloned into the pdTK. Blue/white screening and restriction digestion indicated that recombinant plasmid pdTKUgC was achieved. Thereafter, the downstream arm, digested from pDgC, was inserted into the AsuⅡand KpnⅠsites of pdTKUgC vector, and this generated transfer vector pdgC~-/LacZ~+. The transfer vector contained a gC promoter-controledβ-galactosidase cassette, and there are 25 aa between the ATG start codon (including ATG) and the first amino acid of LacZ gene. Confluent monolayer BT cells were cotransfected with linear pdgC~-/LacZ~+ digested with AsuⅡand IBRV Bartha Nu/67 genomic DNA by the calcium phosphate precipitation technique. Then, the monolayer MDBK cells were inoculated with 1:6 serial diluted virus, which was harvested from the tranfected BT cells with 80％CPE. Overlayed 1％low melting-point agarose and X-gal on the plaque, which were restricted by 1％methyl cellulose before. The recombinant IBRV were picked up from the blue-color plaque, and purified for six generation. The recombinant rlBRV gC~-/LacZ~+ was identified by PCR.Glycoprotein gD, an envelop glycoprotein of IBRV or locating on the surface of the infected cell, induces the highest neutralizing antibody level in the body. In this study, a 943bp fragment was amplified by PCR with the IBRV Bartha Nu/67 genome DNA as template, and was cloned into the pET30a expression vector. Identification by restriction enzyme digestion and sequencing indicated that the recombinant plasmid pET30a-tgD was achieved. Then the recombinant plasmid pET30a-tgD was transformed into E. coli BL21 (DE3). The truncated gD protein (tgD) were expressed in partially soluble form after induction with IPTG, and was purified by immobilized Ni ion affinity chromatography under native conditions. The concentration of purified protein was 0.852mg/ml with high purity (85.2％). Western blot and indirect enzyme-linked immunosorbent assay analysis suggested that the recombinant protein tgD was recognized by IBRV antisera, having a good responsiveness. Therefore, this study is useful for developing diagnosis and subunit vaccine of IBRV.