Cloning And Expression Of Capripoxvirus P32 Gene

Capripoxvirus(CPV) was the agent of goat and sheep pox disease. The CPV structural protein P32 was specific to CPV and has a major antigenic determinant, sequence of P32 gene is also conserved to CPV genome. Diagnosis of CPV infection should be based on the study of P32 protein.Pairs of primers were designed with Primer 5.0 according to Goat pox virus (GTPV) P32 sequence published in Genbank. The templet DNA was separated from scabs of wild CPV infected goat and sheep, and also from cultures of CPV vaccine infected fetal goat dermal cells(FGD-CPV). DNA fragments nearly 1kb were amplified by PCR, and they were inserted into vector pMD18T and sequenced. The results of sequencing show that GTPV P32 gene was 969bp and encode 323 amino acids, Sheep pox virus(SPPV) P32 gene was 972bp and encode 324 amino acids, FGD-CPV P32 gene was 969bp and encode 323 amino acids. The sequences were compared to P32 genes published in Genbank, the results show that the identity between P32 genes is high enough to improve that CPV P32 gene was conserved in CPV genome.The full-length P32 gene was cloned into Bac-to-Bac Baculovirus Expression System and E.coli Expression system. The recombinant expressing vectors were constructed. Bac-Bacl-P32 and Bac-BacHTa-P32 were transfected sf9 cells, pPRO-P32 was transfected E. coli, no targeted proteins were expressed.The smaller truncated P32 fragment without the transmembrane region of P32 was amplified by PCR, and the E. coli expression vector pPRP-TRUNCP32 was constructed. A fusion protein nearly 31kDa was expressed by IPTG-induced BL21(DE3).This assay provided basis for the establishment of rapid and available goat and sheep pox diagnosis methods and for the study of CPV molecular properties.