| Bluetongue(BT)is a severe,economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world.Due to its economic impact,BT is an OIE-listed disease.In China,BTV was first isolated in Yunnan Province in 1979.Since that,at least 20 different BTV serotypes have invaded China,including 31 provinces /cities,BTV1 and BTV16 is the main prevalent serotypes.To establish an effective vaccination program and a DIVA(Differentiating Infected from Vaccinated Animals)test for the control of BT is important.To synthesize BTV16 CLPs,the L3 and S7 genes of BTV16,which encode the two major core proteins VP3 and VP7 of the virus,were inserted into a baculovirus dual-expression transfer vector,and then pFastBac-dual-VP3-VP7 was constructed.By transfected sf9 cells withBacmid-VP3-VP7,the Recombinant baculoviruses named rBac-VP3-VP7 was generated.In the sf9 cells infected by rBac-VP3-VP7,VP3 and VP7 were co-expressed and assembled BTV16 CLPs.BTV16 CLPs were purified by sucrose density-gradient centrifugation.Under the electron microscope,these particles have been observed to be similar to authentic BTV cores.Besides,BTV16 CLPs had the same antigenicity with BTV were confirmed by Western blot tests.The results showed that BTV16 CLPs were constructed successfully,and will be the basis for assembly of virus-like particles and preparation of BTV16 VLPs vaccine.To synthesize BTV16 VLPs,the vector pFastBac-dual was reconstructed to get four multiple cloning sites(MCS)and named pF4.BTV16 VP2,VP5,VP3 and VP7 gene were constructed into pF4.After transfected sf9 cells,a recombinant baculoviruse co-expressing four BTV16 proteins(VP2,VP5,VP3 and VP7)was generated and named rBac-VP2-VP5-VP3-VP7.BTV16 constructive proteins VP2,VP5,VP3 and VP7 were co-expressed and assembled BTV16 VLPs in sf9 cells infected by rBac-VP2-VP5-VP3-VP7.BTV16 VLPs were purified by sucrose density-gradient centrifugation.BTV16 VLPs have been observed to be empty and double-shelled under the electron microscope.In order for reliminary screening of candidate vaccines for BTV16,BALB/c mice were vaccinated by BTV16 VLPs,BTV16 CLPs and BTV16 VP2.The immunogenicity and titer of neutralizing antibody of the VLPs,CLPs and VP2 were tested.The results showed that BTV16 VLPs could induce animal producing high neutralization titres of serum(1:64),but the titer of neutralizing antibody of BTV16 CLPs and BTV16 VP2 were very low.It implied that BTV16 VLPs could be a BTV16 vaccine candidate.For establishing an indirect-ELISA,the gene coding BTV NS3 was inserted into the prokaryotic expression vector pETDuet-1 and expressed in Escherichia coli.Recombinant BTV NS3 protein was purified by Ni-NTA Fast Start Kit.After optimization of the reaction conditions,the indirect ELISA was established,which BTV NS3 was employed as the diagnosis antigen.Then,the diagnosis kit to differentiate the infected and immuned animal of BTV was assembled.The limit of detection of the diagnosis kit can reach 1:1280 dilution of BTV standard positive serum,inter CV is between 3.28% and 9.43%,intra CV is between 3.19% and 12.96%.Compared with IDEXX BTV ELISA kit,it confirmed that the diagnosis kit could distinguish the immuned animals from infected animals of BTV.In this study,the indirect ELISA to differentiate diagnosis the infected and immuned animals of BTV was established,and BTV16 VLPs and CLPs were synthesize byBac-to-BacBaculovirus Expression System.The immune effect implied that VLPs could be a BTV16 vaccine candidate.These results laid a foundation for the diagnostic system and new vaccine research of BT in China. |
