Construction Of Recombinant BHV-1 Expressing The FMDV Structural Protein VP1 And Research Of Immunity

Bovine herpesvirus type 1 (BHV-1) and Foot and mouth disease virus (FMDV) are important cattle pathogens that have a worldwide distribution and are the cause of significant economic losses to the cattle industries. BHV-1 consists of double-stranded linear DNA with an approximate size of 138 kb. Foreign genes can be stably inserted into the genome of BHV-1, which makes the BHV-1 to be a promising live vaccine vector controlling other economically important bovine diseases. Several recombinant BHV-1 expressing immunogenic foreign proteins have been reported as vaccines for other infectious diseases. In April of 2005, an outbreak of FMD caused by serotype Asia 1 began in China. It has been demonstrated that VP1 of FMDV could induce neutralizing antibodies in the experimental and natural hosts. The VP1 is a target protein developing new vaccine for FMD.To express foot and mouth disease virus (FMDV) full VP1 gene and prepare polyclonal antibody against VP1 protein, the VP1 gene was obtained from plasmid pUC57-AsiaVP1 containing VP1 gene of Asia 1 IND 49197 strain by PCR and cloned into the pET-30a(+) for expression in E. coli BL21. SDS-PAGE result showed that protein with a molecular weight of approximately 31.6ku was expressed in inclusion body. The purified recombinant protein showed reactivity to FMD positive serum samples and no reactivity to normal bovine sera in indirect enzyme linked immunosorbent assay (ELISA) and Western blot analysis. These assays demonstrated that the recombinant protein VP1 had very good antigenicity. Antiserum to the VP1 protein was prepared by inoculating rabbits for four times with purified recombinant VP1 protein. The titer of the rabbit anti-FMDV-VP1 serum was 1:20480 in ELISA,1:64 in agar gel immunodiffusion test, and 1:64 in virus neutralization test, respectively. Recombinant protein obtained in this study can be developed to establish a diagnostic test for detecting FMDV Asia 1 antibodies. Polyclonal antibodies can be used to further study the structure, function and epitope mapping of FMDV serotype Asia 1 VP1 gene.In this study, we chose recombinant rBHV-1/gE-/LacZ+acquired previously as parental virus, LacZ gene as inserted site for foreign gene, and constructed recombinant BHV-1 expressing FMDV VP1 gene without reporter gene by way of reversal plaque selection. Firstly, we synthesized plasmid pUC57-AsiaVP1 which contains full FMDV Asia 1 (IND 49197) VP1 gene. Secondly we constructed recombinant transfer vector pGEM T-gE-/AsiaVP1 by substituting LacZ gene of pGEM T-gE-/LacZ+acquired previously for VP1 gene. The vector consists of VP1 gene under the immediate-early promoter of cytomegalovirus(CMV) and BGH polyA, and recombination arms, and without report gene. Thirdly, the mixtures of parental virus genome DNA and transfer vector were cotransfected into bovine turbinate cells (BT) using calcium phosphate-mediated transfection. Finally, the recombinant BHV-1 (rBHV-1/gE-/AsiaVPl) with VP1 gene but without BHV-1 glycoprotein E (gE) gene and LacZ reporter gene was obtained by selection for white virus plaques. PCR results showed that VP1 gene was successfully inserted into the genome of rBHV-1/gE-/AsiaVP1. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting, and the VP1 is stable in serial passage (from1 to 25) of rBHV-1/gE-/AsiaVP1. The 50% tissue culture infective dose (TCID50) of rBHV-1/gE-/AsiaVP1 and rBHV-1/gE-/LacZ+ were 108.43/mL and 108.33/mL respectively, which demonstrated that the distinction of multiplication capacity between the two recombinants was quiet.In this study, we constructed and characterized the immune responses and vaccine efficacy conferred by the rBHV-1/gE-/AsiaVP1. Three groups of adult New Zealand white rabbits each with 5 were inoculated subcutaneously twice 3-week intervals with 1mL 108.33 TCID50 rBHV-1/gE-/AsiaVP1, 1mL 108.33 TCID50 rBHV-1/gE-/LacZ+and control, respectively, and all rabbits were infected with 0.5mL 107.67TCID50 wild virus IBRV/JL06 by the intranasal route 6 weeks post vaccination. The neutralizing antibodies(1:4-1:16) against IBRV could be detected one week post vaccination, and the levels of neutralizing antibodies significantly rose to 1:128 post booster and sustained to the end of this study(eight weeks). There was no difference in the levels of neutralizing antibodies between the two groups vaccinated with rBHV-1/gE-/AsiaVP1 or rBHV-1/gE-/LacZ+. The antibodies(1:256) against FMDV could be detected one week post vaccination by rVP1-ELISA, and the levels of antibodies slightly rose two weeks post inoculation and sustained to the end of this study(eight weeks). The virus was excreted in their nasal secretions for 5 days pi with an average peak of 101.372TCID50/0.1mL or 101.246TCID50/0.1mL on the second day pi, while the non-vaccinated group for 8 days and 102.634TCID50/0.1 mL on the third day pi. The two vaccinated groups showed slightly respiratory clinical signs consisting of labored breathing, conjunctivitis and depression than non-vaccinated group post infection (pi). In conclusion, the rBHV-1/gE-/AsiaVP1and rBHV-1/gE-/LacZ+can protect against clinical disease, which was confirmed by the histological evidence, immunohistochemistry reaction and immunofluorescence assay.In summary, the full VP1 gene of FMDV IND 49197 strain was expressed with prokaryotic expression vector pET-30a (+) in E. coli BL21and preparation of its polyclonal antibodies with rabbits. Moreover, recombinant virus expressing above-mentioned VP1 gene was successfully constructed which could induce rabbits to produce antibodies against FMDV and BHV and protect against clinical disease of BHV. It demonstrated that the newly recombinant BHV-1 rBHV-1/gE-/AsiaVP1 might further be an attractive candidate vaccine for preventing FMDV and BHV-1 simultaneously.