Expression,Purification,and Enzymatic Characteristic Analysis Of Recombinant Bombyx Mori DNA Polymerase ? Complex

As a typical representative of Lepidopteran insects,silkworm(Bombyx mori)is an irreplaceable biological model for the prevention and control of agriculture and forestry pests.silkworm is also one of the important model organisms for studying animal cell differentiation,growth,inheritance and mutation.Silkworm larvae,as a kind of bioreactor,can be utilized to inexpensively and largely produce proteins.In general,eukaryotic DNA polymerase delta is a complex containing many subunits.Mammalian DNA polymerase delta mostly contain four subunits,While insect DNA polymerase delta mostly contain three subunits.Eukaryotic DNA polymerase delta is the most important replicase in the process of chromosomal DNA replication,which has the ability to continuously synthesize DNA.Its 3'-5' exonuclease activity ensures high fidelity of DNA replication,maintains cell cycle normal regulation,the structural integrity and genetic stability of the genome.In this study,silkworm DNA polymerase delta complex was expressed using MultiBac baculovirus/insect cell expression system.The gene sequence of three subunits of silkworm DNA polymerase delta complex was obtained from the silkworm database(GenBank)by bioinformatics comparison with Homo sapiens.Total RNA was extracted from hemolymph of fifth instar larvae of silkworm,and cDNA was obtained by RT-PCR.Bmpol-1,Bmpol-2 and Bmpol-3 genes were obtained by PCR and primers were designed according to the results of bioinformatics comparison analysis.Then,two recombinant plasmids pFastBacHTb-Bmpol-1 and pUCDM-Bmpol-2/3 were prepared respectively.The transposition recombination between pFastBacHTb-Bmpol-1and Bacmid was induced by Tn7 transposition in E.coli cell.The recombinant Bacmid Bmpol-1 plasmid was screened by blue-white screening and resistance screening.The site-specific recombination of pUCDM-Bmpol-2/3 and Bacmid Bmpol-1 at the Cre-loxP site was mediated by Cre recombinant enzyme.Recombinant baculovirus was prepared by transfecting Bacmid Bmpol-1/2/3 recombinant plasmid into Sf-9 cells.RNA was extracted from infectedcells and the Bmpol-1,Bmpol-2 and Bmpol-3 genes were detected by RT-PCR.Western blot analysis showed that BmPOL-1 and BmPOL-3 were expressed in Sf-9cells.poly(dA)/oligo(dT)activity assay showed that BmPOL-1 can not catalyze the synthesis of DNA,whereas Bombyx mori DNA polymerase delta complex have a high effect on DNA synthesis.The target protein was purified using Strep-tactin and Ni2+-NTA affinity chromatography column and the molecular weight of the subunit is123.5 KDa(Bm POL-1),49.1 KDa(BmPOL-2)and 52.6 KDa(BmPOL-3),respectively,determined by mass spectrometry.In summary,silkworm DNA polymerase delta complex with enzymatic activity was successfully prepared by using MultiBac baculovirus/insect cell expression system,which will promote the study of DNA replication and damage repair of silkworm.