LvYY1-mediated WSSV Ie1 Promoter Activation For Enhanced Expression Of Avian Influenza Virus HA In Baculovirus

Baculovirus expression vector system(BEVS)is a eukaryotic expression system developed in the early 1980s.BEVS has been widely used as one preferred platform for the production of recombinant protein complexes and efficacious vaccines.However,for certain antigens,the limited protein yield hinders the application and promotion of BEVS.Therefore,in order to improve the system,the research contents of this study are as follows:1)Exploration of the relationship between the early promoter of white spot syndrome virus(WSSV ie1)and Litopenaeus Vannamei YY1 protein(Lv YY1)in BEVS;2)Identification of the effect of the transcription enhancer Lv YY1 on the WSSV ie1 promoter-mediated transgene expression of reporter gene EGFP;3)Evaluation of the protective immunogenicity of Avian Influenza Virus(AIV)HA expressed in baculovirus modified with Lv YY1-ie1 in chickens.1.Lv YY1 expression and interaction with WSSV ie1 promoter in baculovirusLv YY1-Flag gene sequence was synthesized and inserted into the downstream of Ppolh promoter of p OET5 vector to obtain the recombinant transfer vector p OET5-Lv YY1-Flag.Subsequently,the transfer vector and linearized bacmid were co-transfected to obtain the first generation(P1)recombinant baculovirus BV-Lv YY1-Flag.The recombinant protein was collected and the size of the protein was analyzed in Western blot.The results showed that Lv YY1 was effectively expressed in Sf-9 cells,and revealed a band of 60 k Da.Lv YY1 was further confirmed to be mainly located in nucleus via immunofluorescent assay(IFA).To verify whether Lv YY1 is WSSV ie1 promoter transcription element,gel shift assays were used to examine the interaction of Lv YY1 with WSSV ie1 promoter.Firstly,the recombinant expression plasmid p ET28a-Lv YY1-6×His containing Lv YY1-6×His was constructed and transferred into Rosetta(DE3)E.coli for large-scale expression and purification.Purified Lv YY1 protein was incubated with ie1 promoter sequence(ie1)or the mutated ie1 promoter gene(mie1)amplified by PCR,and then the binding effect of ie1 promoter and Lv YY1 protein was evaluated by gel shift assays.The results showed when incubated with recombinant Lv YY1,ie1 promoter DNA migration was significantly slowed down in a dose-dependent manner,which indicated that Lv YY1could block ie1 movement by interacting with WSSV ie1 promoter instead of mie1.The retarded band migration was also observed when ie1 and mie1 were incubated together in binding buffer for competition assays.To further confirm the interaction in BEVS,nuclear proteins were extracted from Sf-9 cells infected with BV-Lv YY1-Flag and tested as above described.Retarded migration of ie1 promoter was observed in a similar dose-dependent manner.These results indicated Lv YY1 is a specific regulatory element for WSSV ie1 promoter,and its activation relies on specific DNA binding sites.2.Effects of Lv YY1 on WSSV ie1 promoter-mediated reporter gene EGFP expression in BEVSDNA sequences corresponding to ie1-EGFP was subcloned into the p OET5 and p OET5-Lv YY1-Flag vector to obtain the recombinant plasmids p OET5-Lv YY1-ie1-EGFP and p OET5-ie1-EGFP,respectively.Recombinant baculovirus BV-Lv YY1-ie1-EGFP and BV-ie1-EGFP were obtained by transfecting Sf-9 cells.The same titer of recombinant baculovirus was used to infect Sf-9 cells.Emission of green fluorescence was observed at different time points,to analyze the expression level of EGFP.Particularly,BV-Lv YY1-ie1-EGFP presented more and higher intensive fluorescence signals compared with BV-ie1-EGFP.This finding was in consistent with the results in Western blots.WSSV ie1 promoter-mediated expression level of EGFP was significantly increased by Lv YY1.3.Evaluation of Lv YY1-ie1 mediated transgene expression levels in different host cellsTo expand the application of Lv YY1 as transcription enhancer,recombinant virus expressing the main immunogenic gene Hemagglutinin(HA)of H5 subtype avian influenza virus(AIV)was constructed and termed as BV-ie1-HA and BV-Lv YY1-ie1-HA.Sf-9 cells were infected with baculovirus at the same titer to evaluate the level of antigen expression.The results show that supernatant from both baculovirus infected Sf-9 cells showed hemagglutination activity.BV-Lv YY1-ie1-HA displayed a higher mean hemagglutination titer up to 1:512,while BV-ie1-HA developed a mean titer of1:64 at 96 hours-post-infection(hpi).HA yield of BV-Lv YY1-ie1-HA was higher than BV-ie1-HA at the same scale,indicating its advantage in functional HA production.To test the transgenic expression potential of BV-Lv YY1-ie1-HA in target cells,Chicken embryo fibroblast cells(CEFs)were incubated with BV-Lv YY1-ie1-HA at a MOI of 100 for 48 h.IFA analysis revealed H5 specific fluorescent signals were detected in BV-Lv YY1-ie1-HA cells but not in mock cells,indicating the successful transgenic expression in CEFs via BV-Lv YY1-ie1-HA.Moreover,the transduction efficiency of BV-Lv YY1-ie1-HA was verified with q PCR.Significant elevated HA transcription level was detected in transduced CEFs after the incubation with BV-Lv YY1-ie1-HA for 36 h,whereas the mock group only showed a negative transcription level.These results demonstrated that BV-Lv YY1-ie1-HA could act as an attractive vector to deliver protein into CEFs.4.Immunogenicity assessment of recombinant baculovirus BV-Lv YY1-ie1-HATo explore the immunogenicity of newly-developed baculovirus in animal models,14-day-old SPF chickens were randomly divided into six groups(n=5).Group A-C was intramuscularly injected with 200?L different HA titer(2~8,2~6,2~4)of recombinant baculoviruses without adjuvant(BV-Lv YY1-ie1-HA),group D was vaccinated with200?L BV-ie1-HA with 2~4 HA titer,group E was vaccinated with commercial inactivated vaccine as a positive control,and group F was vaccinated with 200?L PBS as a negative control.Sera were collected two weeks after each vaccination for further analysis.ELISA results showed that all different dosages of BV-Lv YY1-ie1-HA vaccinated chickens developed specific antibody response against H5 AIVs.Compared with the commercial inactivated vaccine,BV-Lv YY1-ie1-HA with a titer of 2~8 produced higher levels of neutralizing antibodies,even if was not emulsified.HI results were consistent with the observation in ELISA.Cytokine tests showed that BV-Lv YY1-ie1-HA(2~8 HA titer)could produce IL-4 and IFN-?levels comparable to the commercial vaccine(NS),whereas IL-4 and IFN-?levels increased significantly as compared to the negative control(P<0.05).Based on these results,it was concluded that Lv YY1-ie1baculovirus simultaneously induced strong humoral and cellular immune responses in animal models.