Molecular Cloning And Expression Of DNA Polymerase Gene Of Bombyx Mori Parvo-like Virus (China Isolate)

Bombyx mori parvo-like virus(BmPLV-Z) is a novel virus with two single-stranded linear DNA molecules,VD1(6543 bp) and VD2(6022 bp),which are encapsidated respectively into separate virions.The inverted terminal repeats(ITRs) of BmPLV-Z are imperfect and unable to form palindrome structure.Analysis of the deduced amino acid sequence indicated that VD1-ORF4 encoded a DNA polymerase,which might be involved in the replication of BmPLV-Z.The unique properties of viral genome imply the replication mechanism of BmPLV-Z might be different from that of parvovirus.To elucidate the replication mechanism of BmPLV-Z,an urgent need is to study the function of VD1-ORF4 gene.In this study,VD1-ORF4 gene was cloned and expressed in vitro by pET and baculovirus expression system.The main results were as follows:1.Construction of the prokaryotic expression vector containing the major domains of DNA polymerase geneThe VD1-ORF4 gene is 3318 bp in length,encoding a protein with molecular weight of 120 kDa.It is not conducive to the expression of the full length of VD1-ORF4 in prokaryotic expression system efficiently.In the present study,a 2163-bp fragment was amplified from 3'-terminal of VD1-ORF4 gene.The 2163-bp fragment encoded 720 amino acids, containing two 3'-5' exonuclease motifs and five polymerase motifs of DNA polymerase.The fragment VD1-ORF4/2163 was cloned into prokaryotic expression vector pET-30a.PCR and restriction analysis showed that the recombinant expression vector pET-30a-VD1-ORF4/2163 was constructed successfully.2.Prokaryotic expression of VD1-ORF4/2163 and identification of the recombinant proteinThe recombinant plasmid pET-30a-VD1-ORF4/2163 was transformed into E.coli Rosetta ^TM2(DE3) pLysS.VD1-ORF4/2163 was expressed under the induction of IPTG.SDS-PAGE and Western blot analysis showed that the molecular weight of the expressed protein was about 88 kDa,which was consisted with the caculated molecular weight.MS analysis indicated that the expressed protein was indeed encoded by VD1-ORF4.3.Preparation of polyclonal antibodyExpressed fusion protein was purified by a Ni-NTA column and used to raise polyclonal antibody in New Zealand White rabbit. Harvested antiserum was further purified using protein-A-sepharose CL-4B.SDS-PAGE was performed to analyse the purity of the purified antibody.The result showed that the antibody was purified well.4.Expression of VD1-ORF4 in baculovirus expression system The full length of VD1-ORF4 gene was subcloned into pFastBacHTb.The recombinant Bacmid-VD1-ORF4 was constructed by transposition.Sf9 cells were transfected with extracted recombinant Bacmid-VD1-ORF4 and the supernatants of the transfections were harvested.For expression of VD1-ORF4,a monolayer of Sf9 cells was infected with recombinant virus supernatants.Western blot analysis showed that only a specific band about 120 kDa was detected in the extracts from recombinant virus infected cells,while no similar band was detected in the extracts from wild virus infected cells,indicating that entire VD1-ORF4 of BmPLV-Z has successfully expressed in Sf9 cells.