| BTV belongs to Reoviridae, Orbivirus, Bluetongue virus subgroup, it was spread by Culicoides, purebred landmark alcala gate was the most sensitive animal for this disease, the mortality was as high as35%. The OIE has listed it as A-Class disease and required report it at anytime, Our country provided it as the first class of animal infectious disease, until now, it has no infectivity to human report.BTV was firstly discovered in1876. The outbreak of BTV concentrated in tropical, subtropical and temperate countries; it has brought great losses to the Animal husbandry. In China, it was firstly discovered in1979, until now, it has been separated in29provinces. Until now, the whole world has separated26serotypes of BTV. BTV-1,2,4,9,15,16,23were the mostly serotypes in China, among them BTV-1,and16were the mostly pathogenic serotypes.The genome size of BTV was about19Kb, composed by10parts dsRNA.The genome encoded seven stuctural proteins (VP1to VP7) and four nonstructural proteins (NS1. NS2, NS3, NS3a). VP7protein was encoded by S7gene, composed by349amino acids, it was located on the surface of the virus, VP7protein accounted for36%of the total virus core protein. Relevant research indicated that about94%amino acids of each serotypes BTV VP7protein were highly conserved. The VP7protein was the group-specific antigen of BTV. Grimes etc crystal structure showed that the structure of VP7protein was tripolymer and the function of the top domain (134to253) was antigenic index and combined with the host cell.Since BTV contains so many serotypes and each country has different serotypes, until now there were no effective vaccines to prevent BTV. So, early diagnosis and prevention were the most effective methods to avoid the disease outbreak. The competitive ELISA was the mostly used method in recent years, it is sensitive, specific, stable and safe features, but the imported kit was so expensive, it cannot be applied in animal husbandry. So it seemed imperative that prepared monoclonal antibodies and established Competitive ELISA method by ourselves. In this study, the serotype of BTV-12virus was cultivated by BHK-21cell, and the total RNA were extracted, the VP7gene was amplified by RT-PCR method and cloned into prokaryotic expression vector pMAL-c4X. The recombinant plasmid was transformed into E.coli TB1cells and fusion protein (MBP-VP7) produced after induced with IPTG and purified by resin. Western blot and indirect analysis demonstrated that the fusion protein (MBP-VP7) has favorable antigenicity.In this study, BALB/c mice were immunized by purified MBP-VP7protein, the spleen cells were fused with the SP2/0cells by hybridoma technique, and screened by indirect ELISA coated with purified serotype of BTV-12virus, after3times of sub-clone prepared five strains monoclonal antibody:BTV-1F3,2D10,2H10,4F11and4H7. Subgroup identification results showed that all of the monoclonal antibodies of the heavy chain were IgG1and the light chain were κ. Indirect immunofluorescence test showed that the monoclonal antibodies BTV-2D10and BTV-4H7could occurred specific reaction with24serotypes of BTV but have no reaction with IBAV,CV,AKAV, BVDV, IBRV, BRV, BEV and FMDV. The results indicated that the two monoclonal antibodies were group specific antibodies of BTV.The competitive ELISA test certificated that the BTV-4H7monoclonal antibody has the BTV group specific blocking effect; therefore, the competitive ELISA method was established by MBP-VP7as the coated antigen and BTV-4H7as the competitive antibody. Clinical tests showed the method has100%agreement with IDEXX kit to detect of15positive serum samples and98%agreement with IDEXX kit to detect of322serum samples from Guangxi. Therefore, the competitive ELISA assay established in this study provided a low-cost, effective, quick and accurate method for the detection of antibodies against BTV.The results of M13phage display indicated that the epitopes of BTV-2D10and BTV-4H7were conformational,the epitope of BTV-2D10was decided by four regions at least (185to186,205to207,236to240and278to279), the same that the epitope of BTV-4H7was also decided by four regions at least(34to35,175to177,185to186and278to279). The software discovery Studio combined with the crystal structure which constructed by Grimes etc indicated that the epitopes of BTV-2D10has two resions and BTV-4H7has three resions located on the loop structure of the tripolymer,so they were easily produced antibodies. Compared with all serotypes of BTV VP7genes by the software of DNAStar indicated that the epitopes regions were very conservative in different serotypes of BTV VP7genes and the epitopes of two monoclonal antibodies have two same positions indicated that different BTV Group-Specific monoclonal antibodies have overlapped epitope regions.In order to prove the anthenticity of the M13results, the amino acids of the BTV-4H7epitope were mutanted. In line with nonpolar amino acids to mutate into polar amino acids and exchange of close water type without charge amino acids and close water type with positive and negative charges principles, the amino acids LGIA which were located in the position of33to36were changed to NSKT, the amino acids IFQG which were located in the position of174to177were changed to KKDS, the amino acids MIYL which were located in the position of183to186 were changed to QK.KN, the amino acids WHGL which were located in the position of278to281were changed to KRSN, after that the mutant VP7gene was cloned into prokaryotic expression vector pMAL-c4X, the SDS-PAGE analysis demonstrated that the mutant VP7gene was expressed as same as the natural VP7gene. Indirect ELISA analysis demonstrated that the mutant fusion protein could react with four monoclonal antibodies which producted in this research but could not react with BTV-4H7and the IDEXX’s monoclonal antibody, the results indicated that the epitope of BTV-4H7which displayed by M13was believable and the amino acids which have the effect of block were relative only. These results established the theory basis for the specific blocking mechanism of amino acids which coded the VP7protein. |
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