Construction Of Recombinant BHV-1 Virus Expressing The Major Immunogenic G Genes Of BRSV Virus And Study On Its Biological Characteristics

Infectious bovine rhinotracheitis(IBR),caused by bovine herpesvirus 1(BHV-1),is a disease of domestic and wild cattle?The virus is distributed,The disease is characterised by clinical signs of the upper respiratory tract,such as a(muco)purulent nasal discharge,hyperaemia of the muzzle,dyspnea and genital tract infection,conjunctivitis,Abortion,Mammitis and so on.Bovine respiratory syncytial virus(BRSV)is a pathogen of cattle that can cause disease by itself and also as amember of the bovine respiratory disease complex.As such it is an extremely important bovine pathogen.It has the high infection rate.More than 60%of epizootic respiratory disease in dairy herds is caused by BRSV infection.The impact of BRSV on the economy of the cattle industry is considerable.For this reason,this study use BHV-1/TK-/gG-/gE-as vector to construted the recombinant BHV-1 virus expressing the G gene of BRSV,and research its biological characteristics,security.concrete research contents as Follows:(1)The prokaryotic expression of the BRSV G proteinBRSV G gene was truncated 196-774bp(mG)to connected to pET-3 0a prokaryotic expression vector for constructing the vector pET-30a-mG.The plasmid of pET-30a-mG was transformed into BL21,The mG protein was induced by 1mmol/L IPTG,mG protein expressed efficiently by SDS-PAGE.and the protein was expressed in soluble form.The mG protein was Purified by NI ion affinity chromatography.Western blot showed that the the mG protein has good specificity and reactivity by the caprine origin BRSV positive serum.Rabbits were immunized to make the mG polyclonal antibody,ELISA titration demonstrate the optimum coating concentration of mG protein is 200ng/ml.It is established the foundation to the immunogenicity detection for the recombinant virus in the animal tests.(2)The construction of recombinant BHV-1 virus expressing the major immunogenic G genes of BRSV virus and study on its immunogenicity.In order to construct the transfer vector,The promoter and the multiple cloning site of the PCAGGS was digested with Sal? and Bgl?,And made the promoter and the multiple cloning site connection to the double promoter vector psk-CMV1-BGH-CMV2-SV40,to built the psk-b.actin-BGH-CMV2-SV40 vector.Then the synthetic the G gene of BRSV was connected to the b-actin promoter and the viral genome preparation homology arms of BHV-1 gE was into the transfer vector(pSKBgEpCA.G).A co-transfection of the transfer vector(pSKBgEpCA.G)and viral genome in the MDBK cells was accomplished by lipofectamine 2000,Recombinant virus was purificated by the virus plaque formation technology and identificated by the PCR.Finally,recombinant virus of containing the exogenous fragment G gene was acquired.The fragments of exogenous expression was confirmed by indirect immunofluorescence and western-blot.The exogenous fragment has a good genetic stability through the passaged and PCR identification.In order to evaluate the immune effect of the recombinant virus,these rabbits(1-1.5kg)were inoculated by intranasally with 107 TCID50 of BHV-1/TK-/gG-/gE-/BRSVG+ at avolume of 1 ml,As controls,these rabbits(1-1.5kg)were inoculated with 107 TCID50 of BHV-1/TK-/gG-/gE-at avolume of 1 ml,After the third week,these rabbits were strengthened the immunization again.the blood was collected2?3?5?6 weeks after immunization and the serum was separated.The serum samples was detected by ELISA and neutralization assay,The test results show the BHV-1 neutralizing antibody titer BHV-1/TK-/gG-/gE-/BRSVG+ can reach up tol:32,BHV-1/TK-/gG-/gE-can reach up to 1:30.2.The BRSV mG protein was Coated in the ELISA plates to detect anti-BRSV antibody in serum by ELISA.The results show that it has a certain anti-BRSV antibodies in the serum.Animal experiments showed:Recombinant virus BHV-1/TK-/gG-/gE-/BRSVG+ can induced the antibody against BHV-1 and BRSV in the rabbits.