Generation Of Monoclonal Antibodies Against VP7 Protein Of Bluetongue Virus 14 And Preliminary Establishment Of A Competitive ELISA Detection Method

Bluetongue(BT),an infectious disease of ruminants caused by bluetongue virus(BTV)which belongs to the family Reoviridae and the genus Circovirus,can be transmitted by culicoides and other arthropods.To date,27 serotypes of BTV were identified wordwide.Lack of cross-protection between different serotypes and various clinical symptoms of different serotypes or individuals constitue a challenge for the prevention and control of bluetongue disease and development of polyvalent vaccines.Development of an effective diagnosis kit is great importance for the prevention and control of bluetongue disease.VP7 protein contains group-specific epitopes and can induce immune response,therefore the study of VP7 protein is particularly important.In this study,a strain of BTV-14 was isolated and identified,and consequently VP7 protein was eukaryotically expressed,VP7 specific monoclonal antibodies were generated,and a competitive ELISA detection method was preliminarily established.1.Isolation and identification of BTV-14In this study,the virus was isolated and identified from blood samples collected from individuals suspected of BT infection in a sheep farm.,blood samples were collected from sheep which had suspected bluetongue on a farm,and then subjected to virus isolation and identification.BHK-21 cells were inoculated with samples for blind propagation and cells showing viral cytopathic effects(CPE)were examined with RT-PCR amplification of the S7 and L2 genes,indirect immunofluorescence and neutralization test.The results showed that BTV antibody was positive in some samples,and inoculated BHK-21 cells produced specific CPE and can be specifically recognized or neutralized by BTV-14 standard positive serum.In addition,the S7 gene fragment of 1080bp,the L2 gene of 850 bp and 1581 bp were amplified by BTV respectively.Moreover,amplified L2 gene fragments were purified,sequenced and BLASTed with the NCBI GenBank,it showed that L2 gene sequences matched with BTV-14,This indicates that there is BTV-14 in Xinjiang in China.2.Eukaryotic expression and monoclonal antibody generation of VP7 protein of BTV-14According to the full-length open reading frame sequence of S7 gene of BTV-14,a pair of primers was designed and synthesized.The amplified S7 gene fragment was cloned into pFastBacHTA.The recombinant shuttle plasmid containing S7 gene was collected by transformation into DH10Bac competent cells and plasmid purification,and then was transfected into sf9 cells to express VP7 protein.Meanwhile,BTV-14 virions were collected and purified by sucrose gradient centrifugation and Balb/c mice were immunized according to the immunization protocol.The immunized mouse with the highest titer was selected for final boost three days before cell fusion.The feeder cells were prepared one day before cell fusion and then SP2/0 cells were fused with splenocytes isolated from immunized mice.The eukaryotic expressed VP7 protein was used as a screening antigen,and an indirect ELISA method was established to screen positive clones.A total of 4 hybridoma cells capable of producing antibodies against VP7 protein were obtained.3.Preliminary establishment of a competitive ELISA detection method for VP7 protein of BTThe c-ELISA detection method of BTV was established by using VP7 as the antigen and the prepared monoclonal antibody as a competitive antibody.The optimized reaction conditions were set as follows:plated were coated with the antigen of 1?g/well at 4?overnight and blocked with 5%BSA at 37? for 2 h.The serum to be tested was diluted 5 times,the competitive monoclonal antibody was diluted 400 times,and the enzyme congjuated second antibody was diluted 5000 times.This method is specific and can distinguish FMD(foot and mouth disease)and PPR(small ruminant disease)positive sera samples.Taken together,here a bluetongue virus was isolated and identified as BTV-14 and the S7 gene ORF fragment was amplified,cloned and expressed in sf9 cells.Then,the expressed VP7 protein was used as a screening antigen and 4 positive hybridomas were obtained.Finally,a c-ELIS A method for the detection of bluetongue was established,which laid a foundation for futher developing a rapid diagnosis of bluetongue disease.