Using Baculovirus Expression System Developed Grass Carp Hemorrhage Virus And Avian Influenza Virus Vaccine

The baculovirus expression system has been employed widely as a powerfulexpression vector for the production of recombinant proteins and development ofsubunit vaccines in insect cells.Recombinant baculoviruses containing promoters activein mammalian cells (such as the CMV promoter) can effectively deliver foreign genesinto mammalian cells, resulting in high-level transgene expression, but are incapable ofreplicating in mammalian cells.1. Evaluation of immune efficacy for GCRV vp7/vp6DNA vaccine and subunitvaccineGCRV vp6gene controlled by β-actin promoter (Megalobrama amblycephala)were cloned into the baculovirus transfer vector pFastBacTMDual while the otherstructural protein vp7gene under the control of baculovirus polyhedrin (polh) genepromoter to build expression vector pFastTMDual-VP7-VP6-β-actin contain doublestructural gene under the double control promoter, respectively. It was different formsingle vp6or vp7subunit/DNA vaccine.Bacmid-VP7-VP6-β-actin was obtained andGCRV structural proteins VP7experssion in BmN cells and pupa using the efficientexpression of exogenous genes and efficient gene delivery to vertebrate cells in a safe ofbaculovirus expression system. Cells protein and pupa protein were collectedpost-infectected120h with, the results of SDS-PAGE and Western blotting analysisshowed that structural GCRV VP7protein success expression in specific silkwormtissues. The infected pupae collected at120h post-inoculation with recombinant viruwere used to make freeze-dried powder as an oral vaccine. When the grass carps wereorally adminis trated with feed containing5%of the freeze-dried powder. Results ofRT-PCR showed that the vp6mRNA could be detected in in the blood of oral vaccineimmunization group fish; moreover, VP6protein could be detected in the liver andkidney tissues of orally vaccinated fish and CIK cells infected with Bacmid-VP7-VP6-β-actin.These results suggest that recombinant baculovirus could besuccessfully delivered into the fish.Bac to Bac as eukaryotic expression system in theinsect system can be carried out on the expression of protein modification aftertranslation, therefore synthetic protein has stability and the activity of natural proteinsince its structure is close to the natural conformation.2. Muti epitope antigen protein from avian influenza virus (AIV) wereexpressed in BmN cells using recombinant baculovirus.In order to explore the antigen activity of avian influenza virus (AIV) muti epitopeantigen.In this study,on the basis of silkworm codon preferences and the amino acidsequence of T&B cell epitope box predicted use computer by Cuiqing Zhao to synthesiscorresponding nucleotide sequence, CTLT, THB respectively. The CTLT, THB gene andCMV promoter were cloned into the baculovirus transfer vector pFastBacTM-Dual.Recombinant baculovirus expressing THB&CTLT was generated by site-specifictransposition of the recombinant (pFastBacTMDual-CMV-THB-CTLT) vector into abaculovirus shuttle vector (bacmid) propagated in E. coli DH10Bac cells. Recombinantbaculovirus expressing CTLT was generated by transfecting Bacmid-CMV-THB-CTLTDNA into BmN cells with lipidosome. PCR and Western blot analysis showed thatCTLT expressed in silkworm cells driven by P10promoter.CTLT may be used assubunit vaccines after infection the recombinant virus while the virus DNA may be usedas THB nucleic acid vaccine.