Screening Of Proteins Interacting With AcPCNA And An Improved Method For BmNPV-POL Purification

Autographa californica multinuclear polyhedrosis virus(AcMNPV) and Bombyx mori nuclear polyhedrosis virus(BmNPV) have been considered as the model virus of baculovirus. The study on their replication mechanism always attracts the attention of the domestic and foreign researchers. In the study of the replication mechanism in eukaryotes, proliferating cell nuclear antigen(PCNA) was found to improve polymerase processivity which played a fundamental role actting as DNA polymerase δ cofactor. The biological phylogenetic analysis of PCNA found that only nine kinds of baculovirus encode their PCNAs. However, their specific functions have not been known exactly.It had found that AcMNPV genome encoded a homologue of the PCNA which had been named AcPCNA. However, AcPCNA was not essential for viral DNA replication based on analysis using a transient replication assay. In order to further reveal its function, in this study, recombinant prokaryotic expression plasmid pET30a-AcPCNA was constructed successfully based on the gene sequence of AcMNPV PCNA provided by Gen Bank. Meanwhile, the pET30a-AcPCNA was transferred into E.coli BL21 and expressed by IPTG induction. The interested protein was identified by Western Blot. A large amount of His-AcPCNA was obtained by using Ni-NTA column density gradient elution and Mono Q ion exchange chromatography. At the same time, SuperoseTM 6 gel filtration results showed that the molecular weight of AcPCNA was about 100 kDa under non-denaturing conditions, whereas the predicted molecular weight was 30.7 kDa. It is shown that AcPCNA was likely to express and assemble with a trimeric form. The purification AcPCNA elution fractions were coupled with CNBr-activated Sepharose 4B to prepare an affinity chromatography column after ultrafiltration concentration. The total protein of Sf-9 cells infected by wild type(wt) AcMNPV which was sonicated and high speed centrifugated at low temperature, flowed through AcPCNA coupling affinity chromatography column. Twelve kinds of suspected AcPCNA interacting proteins were identified by mass spectrometry. These included the Sf-9 ribosomal composition protein(Ribosomal protein L3), Actin protein which were associated with protein synthesis and LEF3, p48 protein and other proteins of AcMNPV. Affinity chromatography conjuncted with mass spectrometry to find AcPCNA interacting proteins is very significant for understanding the virus DNA repair mechanism, which will lay an important biological foundation for further research.In order to study the influence on BmNPV-POL activity of AcPCNA after translation modification by eukaryotic expression system, the pFastBacHTb-AcPCNA was constructed and transfered into DH10 Multibac competent cells and obtained the recombinant Bacmids by blue-white selection in this study. After that, the recombinant viruses were prepared by transfection Sf-9 cells. And eukaryotic expressed AcPCNA influence on polymerase activity of BmNPVPOL was analyzed in Poly(dA)/Oligo(dT) analysis. But it did not find the stimulate activity. It indicated that BmNPV-POL was AcPCNA independent.Bombyx mori nuclear polyhedrosis virus DNA polymerase(BmNPV-POL) encoded by BmNPV ORF53 plays an important role during the process of baculovirus replication process. Failure to obtain sufficient quantity of the high-purity active protein has limited the research on BmNPV-POL. This study reports the one-step purification of BmNPV-POL by means of affinity chromatography on Strep Tactin. This was achieved by designing specific primers PCR to replace the His tag with strep tag affinity peptide in C-terminus of the pFastHTc-BmNPV-POL-His recombinant vector which had been built in the early study. Target protein expression and enzyme activity were detected by Western Blot and Poly(dA)/Oligo(dT). The results showed that it owned a high enzyme activity. High-purity active BmNPV-POL was purified by one step purification through Strep Tactin affinity chromatography column, which could provide adequate material for subsequent experiments. In summary, this simple method decreased protein loss and improved the mass production of BmNPV-POL, which will promote research and development of BmNPV-POL in the future.