The Expression Of The PLA2 Domain Of The Periplenta Fuliginosa Densovirus Structural Protein Gene In E. Coli And Preliminary Study Of Its Function

Periplaneta fuliginosa is the major species of cockroach distributed in urban and rural area of our country. Periplaneta fuliginosa Densovirus(PfDNV) is the first classified and authenticated cockroach densovirus. Although the research on the biophysical properties, genomic structural analysis and Cytopathology of the virus have been accomplished, the function of structural protein and the molecular mechanism of its infection are still unknown in case of the absence of the stable cell model of PfDNV infection in vitro.Recently it was reported that a phospholipase A2 motif was discovered in the structural protein sequences of parvovirus. The parvovirus phospholipase A2 motif shared amino acid homology with the secreted phospholipase A2 motif(eg, snake venom and bee venom), including the conserved calcium binding loop YXGXG and the catalytic helix site HDXXY. Also, The insertion and deletion experiment has showed that the function of this PLA2 domain was related to infection of virions.So, in order to get further information ,we study the PfDNV phospholipase A₂ functional domain in protein level. The fragment was obtained by PCR amplification. The amplified products was ligated with pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The in-frame sequence confirmed recombinant plasmid pET28a-PLA and pET26b-PLA were transformed into E. coli BL21-codonplus-(DE3) -RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion proteins were produced. By optimazing the expression condition, we get soluable fusion protein in cytoplasm and periplasm through the induction of pET26b-PLA plasmid. The target fusion protein was purified with Ni-NTA affinity column, then analyzed the PLA2 enzyme activity using the substrate phosphatidylcholine ,the results demonstrated that the recombinant PLA2 protein of PfDNV was successfully expressed, which would facilitate the preparation of the antiserum and the further study of the biological properties of PLA2 enzyme.