Cloning And Expression Of P32 Gene Of Goat Pox Virus

Goat pox is an acute and highly contagious viral disease of goats and sheep, characterized by generalized pox lesions throughout the skin and mucous membranes, a persistent fever, lymphadenitis, and often a focal viral pneumonia with lesions distributed uniformly throughout the lungs. The pathogen of this disease is goat pox virus. In adult goats, morbidity may range to 80 percent and mortality can approach 50 percent. In susceptible kids under 1 month of age, morbidity may approach 100 percent, and mortality may be as high as 95 percent. Recently, this disease is epidemic and broke out in many areas in China and caused serious damage to the goat husbandry. However, to date, there is no specific medicine to cure this disease, and the best way to control this disease is prophylactic immunization of all susceptible animals with a potent and efficacious vaccine. Consequently, it is much more important to make early and proper diagnosis and then prevent the disease from prevailing. The protein P32 is an envelope protein which is specific to the members of Capripoxvirus genus and has a good antigenic determinant. Compared with other proteins of goat pox virus, P32 could induce body to produce higher level antibody quickly after inoculation. This protein could be used as antigen to set up a set of diagnostic method and also could be used as subunit vaccine.In this experiment, the goat pox virus genome DNA was extracted from the goat pox virus attenuated vaccine. According to the nucleotide sequence of p32 gene published in the GenBank, a pair of primers was designed and synthesized to amplify the full length p32 gene. Then the p32 gene was cloned into the vector pMD18-T and sequenced. The result of sequencing shows that GPV p32 gene is 969bp long and codes for 322 amino acids. The sequence of P32 was aligned with the sequences of P32 published in the GenBank. The results show that the nucleotide identity to p32 of other strains of Capripoxvirus is 97.5%-99.9% and the amino acid identity is 95%-99.7%, which indicates that p32 gene was conserved in all strains of Capripoxvirus. Software TMHMM predicts that P32 has a transmembrane domain between the amino acids 281 and 303.After cloning the p32 gene into the vector pMD18-T, p32 gene was subcloned into the expression vector pET-30-a(+),pGEX-6P-1 and pPROEX^TMHTb respectively. Then we transformed those recombinant plasmids into E.coli Competitive Cells BL21(DE3) or DH5 α and induced them to express the target protein using IPTG. Based on analysis by SDS-PAGE, it appeared that target protein was not expressed at a high level. Considering the bad expression results in Prokaryotic Expression System, Bac-N-Blue^TM baculovirus expression system was utilized as an alternative way. The p32 gene was subcloned into baculovirus transfer vector pMelBacA to construct the recombinant plasmid pMelBacA-p32. The pMelBacA-p32 was identified by digestion with restriction enzymes and by PCR using primers specific for p32 gene. The pMelBacA-p32 was purified and co-transfected into Sf9 insect cells with linear baculovirus DNA (Bac-N-Blue^TM DNA) by the technique of cationic liposome mediated transfection. The recombinant baculovirus was purified by screening for blue plaque three times, which was named as rBaculovirus-p32. Baculovirus genome DNA was isolated and amplified with primers specific for p32 gene and recombinant baculovirus respectively. The result indicates that recombination...