Construction Of A Highly Efficient Display System For MultiBac And Its Application On Multigene Combined Vaccine

Baculovirus display system(BDS) is an excellent eukaryotic surface display system with the advantages of safety,efficiency and economy.Baculoviral vector is widely used in antigen presentation,gene therapy and genetic engineering vaccines due to their high safety,high efficiency gene expression level and better protein modification.Recent researchs have indicated that BDS is mainly limited by the following,(1)The fusion expression of target gene and Class ? envelope gene are used to realize the envelope display of target protein in traditional BDS,while the baculovirus also expresses the wild-type envelope protein with high-level,which leads to strong competition between the two envelope proteins,resulting in low efficiency of the target protein display;(2)The lack of transfer vectors and recombination sites for foreign genes,resulting in BDS only focuses on single-or dual-target proteins,while the studies on fusion of multiple proteins has not been explored;(3)The expression of the fusion gene by the immediate late promoter of the virus in traditional BDS,generally.The time difference in expression of the promoter also causes competition between the proteins and reduces the display efficiency.Furthermore,the baculovirus promoters have typical viral protein-dependent characteristics,which does not have expression activity in most non-host cells;(4)Although the envelope protein could mediate the recombinant baculovirus transduction to mammalian cells,which the inefficiency resulting in their inability to achieve the application of BDS.Consequently,improving the display efficiency of BDS,exploring the co-display ability of multi-target proteins,screening a high-efficient promoter independent of transcription factors in host cell,and improving the transduction efficiency of baculovirus to non-host cells,which are the major problems that need to be solved in the development of BDS.In this study,the homologous recombinant gene gp64F-Zeocin^R-gp64R containing Zeocin resistance gene(Zeocin^R) was screened and designed,and the gp64 gene deletion type E.coli AcMulti Bac/?asd Sw106/PGB₂?Inv/?gp64(Sw106-?gp64)was constructed by Red-gam recombination.At the same time,the deletion of the envelope GP64 based on Sw106-?gp64 determined that the recombinant baculovirus could not complete the viral budding and maturation process.Subsequently,the expression of the luciferase mediated by ires(Internal Ribosome Entry Site)was lower than that upstream promoter in insect Sf9 cells significantly,and the maximum difference in expression can reach 0.25 times(12 h.p.i,p<0.01).Further studies showed that the ires could significantly reduce the expression of GP64 and rescue the replication cycle of gp64 deleted baculovirus.Transmission Electron Microscope(TEM)also showed that low-GP64 assembly had no significantly effect on the envelope structure and viral morphology of recombinant virus particles.In order to reveal the enhancement of display efficiency and display ability by ires-mediated low-level expression of GP64,the results of fusion display of luciferase and fluorescent protein based on Sw106-?gp64 showed that ires-mediated low-level expression of GP64 increase the display efficiency of Luciferase about five times(p<0.01).The results of Laser Scanning Confocal Microscope(LSCM) and Western Blot indicated that Zero-Background Tn7 and Cre-lox P specific transposons successfully achieved stable and efficient co-display of three fluorescent proteins(GFP,m Cherry and YFP)on the membrane surface of the sf9 cell and the envolpe of the recombinant virus.According to the expression efficiency of baculovirus promoters(p10,p64,pvp39)and mammalian cell promoters(pcmv,psv40,pie1) in sf9,BHK-21 and 293T cells at different time,an immediate early active promoter p64 and non-viral protein dependent promoter pie1 were screened successfully to mediate the efficient expression and display of fusion genes in BDS system.In addition,flow cytometry demonstrated that the modification of Invasin and VSVG on envelope could significantly improve the transduction efficiency of recombinant baculovirus to non-host cells(BHK-21,293T).Based on the above results,the fusion display recombinant baculovirus with high efficiency and four target proteins(PCV2-Cap,PRRSV-Gp5,CSFV-E2,Invasin)was successfully constructed by Sw106-?gp64 and ires-mediated low-expression of GP64.Western Blot and envelope antigen ELISA indicated that the display efficiency of the four target proteins was significantly higher than traditional BDS(p<0.01).The immunoassay further confirmed Sw106-?gp64 and ires-V5gp64 compensatory expression,which resulted in higher levels of specific antibodies in BALB/c mice than traditional BDS(p<0.01).To summarize,this study successful constructed a gp64 gene-deficient E.coli Ac Multi Bac/?asd Sw106/PGB₂?Inv/?gp64 successfully.Through ires-mediated low-level expression of GP64,the envolope display efficiency and display stability of multi-target proteins were improved significantly,which do not affecting the replication cycle and virus morphology of the recombinant virus.Secondly,high-expression of fusion gene mediated by baculovirus immediate early active promoter p64 and viral protein-independent promoter pie1 was successfully achieved by promoter expression sequence.Futhermore,modification of the viral envelope by Invasin and VSVG could further improved the transduction efficiency of the recombinant virus to non-host cells.Finally,based on the above research,a novel recombinant baculovirus with high efficiency of multi-target protein co-display was constructed and successfully applied to mouse model.The development prospects of Sw106-?gp64-ires-V5gp64 in multi-protein co-display,multi-vaccine and other fields were systematically explored,which laid a foundation for the development of BDS.