Journal of Immunology and Immunopathology

Bluetongue (BT) is an infectious non-contagious viral disease of small ruminants. The structural protein VP7, encoded by RNA genome segment 7, is major group specific antigen. This protein is capable of detecting antibodies irrespective of serotypes and thus has potential in the detection of all BTV serotypes. Keeping in view the diagnostic importance of the VP7, the complete and truncated (from nucleotide position 321–1070) VP7 gene were amplified by RT-PCR and cloned in prokaryotic expression vector, pPROEX HT and eukaryotic expression vector, pBK-RSV. The expression in E. coli was induced with 1 mM IPTG. No expression could be noticed in the clone containing complete VP7 gene (pPRO-EX-a.VP7.bt) whereas, protein 90 kDa alongwith expected 30 kDa protein were expressed in the clone containing truncated VP7 gene, pPROEx-b-770.bt. These proteins were confirmed to be BTV specific by western blot analysis using anti BTV sheep serum, mono specific VP7 serum raised in rabbit after DNA immunization and serum raised in rabbits against multiple antigenic peptide (MAP) chemically synthesized using amino acid sequences from VP7. Transfection of the MDBK cells with pBK-RSV-VP7.bt plasmid DNA resulted in expression of 38 kDa protein, authenticity of which was verified by western blot analysis. Immunization of rabbits with this construct resulted in induction of bluetongue virus specific antibody which was reactive to virus in ELISA, dot-ELISA and AGPT. Use of these clones in bluetongue diagnosis has also been discussed.