Assembly Of Bm CPV Virus VLPs Based On Multi Bac Expression System

Bombyx mori cytoplasmic polyhedrosis virus belongs to reovirus type polyhedrosis virus.The three-dimensional reconstruction of cryo electron microscopy based on near atomic resolution shows that BmCPV is a symmetric positive 20 surface body with 12 protrusions on the vertex of the capsid,and the viral capsid is composed of 60 asymmetric units,each of which consists of turret protein,large protrusion protein and capsid shell protein.BmCPV has no capsular membrane.The characteristics of single shell make it a model material for three-dimensional reconstruction of cryo electron microscope.In this study,the CSP and LPP of BmCPV were used as the target protein to construct the transfer vector pU-CSP-His-mCherry and pF-LPP-His-eGFP,and the transfer carrier pUCSP-His-mCherry was reorganized into AcMultiBac by using the homologous Cre-Loxp locus,and the transfer carrier pF-LPP-His-eGFP was transferred to AcMultiBac by Tn7 transfer to the seating point.Ac-rSw106-inv-mCherry,Ac-rSw106-inv-LPP-eGFP and AcrSw106-inv-CSP-mCherry-LPP-eGFP recombinant bacteria solution.Sf9 cells were infected with Ac-rSw106-inv-CSP-His-mCherry,Ac-rSw106-inv-LPP-eGFP and Ac-rSw106-invCSP-mCherry-LPP-eGFP recombinant strains expressing the infection protein.After 3 days,cherry red fluorescence,enhanced green fluorescence and co expression of two kinds of fluorescence were observed respectively.The recombinant virus Ac-CSP-His-mCherry,AcLPP-His-eGFP and Ac-CSP-His-mCherry-LPP-His-eGFP were collected from the supernatant,and Western blot was used to detect the relative molecular weight of the target protein,which proved that CSP and LPP were expressed successfully in Sf9 cells.The Sf9 cells infected by recombinant virus Ac-CSP-His-mCherry and Ac-CSP-His-mCherry-LPPHis-eGFP were broken and purified by sucrose density gradient centrifugation,and two different VLPs,CSP-VLPs and CSP-LPP-VLPs were obtained.Under transmission electron microscope,two good forms of VLPs were found.The physicochemical properties and homology modeling of CPV virus structural protein provide a theoretical basis and reference for the expression and purification of recombinant protein.At the same time,through bioinformatics analysis of CPV virus structural protein CSP and LPP codon preference,it is found that the above two structural proteins are suitable for the expression of Sf9 cells,but the use of codons has certain differences,which provides a codon optimization scheme for improving the expression of CSP and LPP in subsequent experiments.The expression of CSP-VLPs and CSP-LPP-VLPs by the rod like polygene expression system provides a good theoretical basis for the in-depth analysis of the structure of the shell virus.At the same time,the construction of a good CSP-LPP-VLPs can carry foreign molecules inside and outside.It can be used for gene therapy and delivery tools,the development of new biological pesticides,and the preparation of new nanoscale materials,transport RNAi,display epitope peptides and other applications.It has a good prospect.