Study On The Recombinant Baculovirus Incorporating A Hybrid CMV Promoter/SFV Replicon

The Baculovirus Autographa californica multiple Nucleopolyhedrovirus(AcMNPV) has long been used as a biopesticide and as a tool for high-level expression of recombinant protein in insect cells.Its host specificity has been considered to be restricted to cells derived from arthropods,However,Hofmann et al.,first in 1995 reported that recombinant baculovirus containing cytomegalovirus immediate-early(CMVoIE) promoter can drive the expression of a luciferase reporter gene in human hepatocytes. Since this initial report,the baculovirus/mammalian expression system has been employed with different mammalian cell-active promoters,such as Rous sarcoma virus (RSV) promoter or a hybrid CAG promoter that constituted a CMV-IE enhancer, chickenβ-actin promoter,and rabbitβ-globin polyadenylation signal,to deliver genes into numerous mammalian cells.Alphavirus,including Sindbis virus,Semliki Forest virus(SFV),and Venezuelan Equine encephalitis(VEE) virus,has also catched considerable attention for use as expression vectors.The significant characterizations of alphavirus vector are self-amplification,high-level expression ofheterologous protein,and the induction of apoptosis in the infected cells.The key component of alphavirus for these propertes lies in its RNA "replicon",the RNA replicase complex,which is encoded by the viral nonstructural genes(nsPs) and is required for replication of the genomic RNA and transcription of subgenomic RNA.In this report,we constructed a baculovirus vector containing the SFV replicon under the transcriptional control of CMV-IE enhancer/promoter,enhanced green fluorescence protein(EGFP),which is immediately under the SFV 26S subgenomic promoter,was generated to evaluate the gene delivery efficiencies and heterogenous protein expression in mammalian cell lines.The most research works were as following:1.Construction and expression of recombinant baculovirusThe DNA fragment containing CMV-IE enhancer/promoter-SFV replicon-26S promoter was released from pSCA1(kindly provided by Dr.Bremner,University of Toronto,Canada) with SphI and SpeI digestion,and inserted into the pFastBac DUAL (Invitrogen) backbone to replace the pl0 and ployhedrin(pPolh) promoters of Baculovirus,resulting in pFastBac-CMV/SFV.EGFP reporter gene was obtained from pc-EGFP by BglⅡand BamHI digestion and then inserted into the unique BamHI site of pFastBac-CMV/SFV,immediately downstream of SFV subgenomic promoter 26S,to generate pFB-CMV/SFV-EGFP.The DNA fragment containing CMV-IE enhancer/promoter-EGFP was obtained from pc-EGFP by BamHI and NotI digestion and then inserted into pFastBacl to generate pFB-CMV-EGFE The recombinant baculoviruses were subsequently generated by using the Bat-to-Bat(?) system(Invitrogen) following the manufacturer's instructions.The virus was further amplified by propagation in Sf-9 cells.By compared with Bac-CMV-EGFP,Bac-CMV/SFV-EGFP hereafter is a recombinant baculovirus,capable of expressing the reporter gene,an enhanced green fluorescence protein(EGFP) under control of this hybrid promoter,exhibiting high transduction efficiency and high-level expression of reporter protein in mammalian cells. Optimal transduction conditions,including transduction temperature,time and dose, were also investigated.Additionally,the recombinant baculovirus can induce apoptosis in mammalian cells in the course of transduction,demonstrated by observed DNA laddering patterns and increased caspase-3 activity.2.Construction and immune effect of modified recombinant baculovirusAlthough AcMNPV are failing to replicate in vertebrate cells,it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene.Follwing these findings,baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells.However,in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system.Therefore,in this study we describes the generation of a recombinant baculovirus in which the vesicular stomatitis virus glycoprotein G(VSV-G) is present in the viral envel6pe.We inserted the SV40 fragment into the plasmid pFastBac-G,which in order to offer the restricted enzyme sites for the SFV replicon,to generate the plasmid pFastBac-G-SV40,and then the DNA fragment containing CMV-IE enhancer/SFV-EGFP was released from pSCA-EGFP with SphI and SpeI digestion,and inserted into the pFastBac-G-SV40,resulting in pFB-G-CMV/SFV-EGFP.The recombinant baculoviruses were subsequently generated by using the Bac-to-Bac(?) system(Invitrogen) following the manufacturer's instructions. The virus was further amplified by propagation in Sf-9 cells.To characterize the induction of antigen-specific immune response mediated by baculovirus,mice were subjected to intramuscular with baculovirus,ELISA analysis showed that relativatly hifher EGFP-specific antiboby was detected in mice immunized with Bac-G-CMV/SFV-EGFP after the primary immunization.Following a boost at week 3,' antibodies increased significantly and the mean ELISA antibody level was reach to 1:1440,In agreement with the humoral immune response,at 6 weeks after primary immunization,the highest celluar immune response was found in restimulated splenocytes from mice immunized with Bac-G-CMV/SFV-EGFP. 3.Construction and immune effect of recombinant baculovirus expressing the HA geneFurther more,HA gene was employed to construct the recombinant baculovirus Bac-G-CMV/SFV-HA.Expressed HA protein was confirmed with indirect immunofluorescent assay on Mammalian cells,mice were subjected to intramuscular with this baculovirus,compared with the plasmid pSCA-HA.The result showed that the recombinant baculovirus Bac-G-CMV/SFV-HA was as good as plasmid pSCA-HA on the humoral immune response,however,the recombinant baculovirus was much more better than pSCA-HA on the celluar immune response.In summary,the recombinant baculovirus/mammalian vector described in this study combines the safety and high infectivity of the baculovirus vector with the high level of foreign gene expression and the proapoptotic properties of SFV replicon,which will facilitate the development of baculovirus/mammalian gene delivery system for future gene therapy and vaccine researches.