Construction Of A Recombinant BHV-1 Expressing VP1 Gene Of FMDV And Its Immunogenicity In Rabbits

Infectious bovine rhinotracheitis (IBR) is a widespread viral disease of cattle that causes serious economic losses in the cattle industry worldwide. The causative virus is bovine herpesvirus type 1(BHV-1), commonly known as infectious bovine rhinotracheitis virus, a member of the Herpesviridae family, alpha herpesvirinae subfamily. BHV-1 consists of double-stranded linear DNA with an approximate size of 135 kb, and foreign genes can be stably inserted into the genome of BHV-1, which makes the BHV-1 a promising candidate for the development of a live vaccine vector for economically important bovine diseases. Several recombinants expressing immunogenic foreign proteins have been reported as vaccines for other infectious diseases.Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious and economically important disease affecting cloven-hoofed animals. FMDV consists of a single-stranded, positive-sense RNA genome of approximately 8,500 bases surrounded by four structural proteins, VP1, VP2, VP3 and VP4. It has been demonstrated that VP1 could induce neutralizing antibodies in the experimental and natural hosts and also involved in the interaction between virus and cell surface receptor. So the VP1 is a target protein for develop new vaccine for FMD. This research was aimed at constructing a recombinant BHV-1 expressing VP1 gene of FMDV as a candidate vaccine strain to develop a novel bivalent vaccine against FMD and IBR.In order to construct the recombinant BHV-1 which expressing FMDV VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV (O/China/99) under the immediate-early promoter of cytomegalovirus. The mixtures of parental virus (BHV-1/gE-/LacZ+) DNA and transfer vector were cotransfected into bovine turbinate cells using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 (designated BHV-1/gE-/VP1) was obtained by selection for white virus plaques. PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE-. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting.In order to express foot and mouth disease viru(sFMDV)(O /China /99)VP1 gene and prepare the VP1 protein polyclonal antibody for providing a basic materials for identification of VP1 expression in cells infected BHV-1/gE-/VP1, the VP1 gene was amplified by PCR from the plasmid pUC57-VP1 and cloned into the prokaryotic expression vector pET-30a(+). the recombinant plasmid pET30a-VP1 was transformed into E.coli BL21 after confirmation and then induced with IPTG, SDS-PAGE result showed that a protein approximately 38ku was expressed in inclusion body. Western blot and indirect ELISA results showed that recombinant protein can react with FMD positive serum. The BALB/c mice were immunized with pirmarily purified recombinant protein to prepare polyclonal antibody againist the VP1.To compare the multiplication capacity between BHV-1/gE-/VP1 and BHV-1/gE-/LacZ+, the 50% tissue culture infective dose (TCID50) was calculated, they were 107.9/ml and 107.3/ml respectively. To analyze the genetic character of BHV-1/gE-/VP1, 5,10,15 passages of BHV-1/gE-/VP1 DNA were taken as templet for PCR, the result showed that VP1 gene can be amplificated. Eight New Zealand white rabbits were randomly divided into two groups, the rabbits were inoculated subcutaneously with 1 ml 1×10^7.0 TCID₅₀ BHV-1/gE-/VP1 and 1 ml 1×10^7.0 TCID₅₀ vector virus respectively. The second inoculation was given with the same dose at two weeks post-inoculation and the blood samples were obtained once a week, and sera were collected to determine the presence of antibodies against IBRV and VP1 protein. The immunogenicity was confirmed in a rabbit model by virus neutralization test and ELISA.We successfully constructed BHV-1/gE-/VP1 which expressing foot and mouth disease virus VP1 gene. BHV-1/gE-/VP1 had similar multiplication capacity compared with BHV-1/gE-/LacZ⁺ and had stable genetic character. And the immunogenicity was confirmed in a rabbit model. This research provided a technical platform and basis for applying BHV-1 as vector for development recombinant vaccine of expressing FMDV VP1 gene and other foreign genes.