Prokaryotic Expression Of Bluetongue Virus VP7 Protein, Schmallenberg Virus N Protein And Development Of Indirect ELISA

Bluetongue(BT) is a potent non-contagious disease, which was caused by the bluetongue virus(BTV). The virus belongs to bluetongue virus subgroups of Obivirus of Reoviridae, which mainly occurs in sheep. BTV is double-stranded RNA virus with 10 fragments(segment 1~segment 10) encoding seven structural proteins(VP1~VP7) and five non-structural proteins(NS1, NS2, NS3, NS3 a, NS4). VP7 protein is encoded by S7 gene, which carries group-specific epitopes and contains at least six linear epitopes. The amino acid sequences of different serotypes of BTV VP7 proteins were analysised using DNAStar software. The results showed that: the homology of 30 to 48 amino acid residues sequence is 100% among all sequences of different serotypes of BTV VP7 proteins. This study proves that VP7 protein is a highly conserved protein with group-specific, and is ideal antigen for the detection of antigen-antibody BTV.Schmallenberg disease is a new type of viral disease, caused by Schmallenberg virus(SBV). SBV is single-stranded negative-strand RNA with three fragments, and belongs to Simbusero group viruses of Orthobunyavirus of Bunyaviridae. This disease spread to most of Western Europe region, which caused huge losses to the farming industry. This disease has not emerged in China. The nucleocapsid N protein is the strongest antigen protein of Schmallenberg virus-encoded protein.To illustrate the immunogenicity of VP7 protein of bluetongue virus(25 type), VP7 gene was amplified and cloned in pET-24b(+) expression vector. The pET-24b-BTV-VP7 recombinant plasmid was transformed into BL21(DE3) competent cells, then the VP7 protein of bluetongue virus was expressed and purified.The results showed that, the molecular weight of VP7 protein is 40 KD, which has good immunogenicity. The indirect ELISA was developmented,which optimization of reaction conditions shows, 80-fold dilution of antigen, a 400-fold dilution of positive serum, 5000-fold dilution of HRP-conjugated AffiniPure Donkey Anti-Goat IgG are the optimal dilution. We compare ELISA method to a commercial kit IDEXX simultaneous. The result is almost same. In order to identify the specificity, VP7 antigen is coated to detect other 4 positive sera. The results show that the cross reaction did not happen with the other four kinds of virus. We indicate that the method can be used to detect the epidemiology of BTV, at the same time the operation is simple and the cost is low.Because of Schmallenberg virus is a foreign virus, and the disease has not yet appeared in China. According to GenBank nucleotide sequence(BH80/11-4 SBV), the N protein sequence was synthesized artificially. To illustrate the immunogenicity of N protein of Schmallenberg virus, N gene was amplified and cloned in pET-24b(+) expression vector. The pET-24b-SBV-N recombinant plasmid was transformed into BL21(DE3) competent cells, then the N protein of Schmallenberg virus was expressed and purified. The indirect ELISA was developmented by N protein(antigen) specifically reacting with monoclonal antibodies. 640-fold dilution of antigen, a 100-fold dilution of monoclonal antibodies, 5000-fold dilution of HRP-conjugated AffiniPure Donkey Anti-Mouse IgG are the optimal dilution. and under the same conditions, to detect other 4 virus-positive serum in order to identify the specific method. The results showed that the monoclonal antibody specifically reacts to purified N protein, and between four kinds of different virus positive serum and N protein does not react. This method indicats indirect ELISA method had good immunogenicity.