Expression Of Shev ORF2Gene In Bombyx Mori Baculovirus Expression Vector System

【Objective】As there is not effective vaccine for HEV at present，this study screened virus strainhigh expressing HEV-ORF2in Bombyx mori baculovirus expression system and establishedfoundation for diagnostic reagent of antigen or antibody and safe genetic engineering vaccine.【Method】(1)To study the antigenicity of Prokaryotic expression of main antigenic area in TheC-terminal of HEV-ORF2gene,The C-terminal of the ORF2gene of hepatitis E virus (HEV), whichcontains289amino-acid residues, was amplified with PCR and sequenced. We constructed therecombinant with the vector pET-28a(+), and expressed in E.coli BL21(DE3) by0.5mmol∕L IPTGinduction at different temperature. The result of ELISA showed the protein had good antigenicity.(2)In this experiment,we selected procusor SHEV of SW189strain,which were inserted intoBombyx mori baculovirus transfer vector pVL1393and constructed the recombinant vector, withwhich co-transfected linearted DNA of Bm-BacPAK6virus into BmN cells. The recombinant viruswas gained. Indirect immunofluore-scence test was determined that recombinant virus could validlyexpress the target gene in Bm-N cell. At the same time, the fifth instars were infected with therecombinant virus. The target protein in haemolymphe was expressed according to indirect ELISAresult. Screening and purification of recombinant virus, with which the fifth instars were infectedagain. The hemolymph collected was detected by the ultra-centrifuged thereafter. The result ofelectron microscope indicated that the HEV antigen has been highly expressed in Bombyx moribaculovirus expression system.【Results】(1) Target gene was cloned protokaryon expression system correctly by double digestand sequencing；By screening expressing conditions,the highest expression was with0.5mmol/LIPTG inducing4h，37℃.(2) Recombinant protein had antigenicity and expressed right by MSanalysis；antiserum titer reached1:3200and met the requirements for eukaryon expression detection.(3) The hemolymph detection after recombinant virus infecting Bombyx mori indicated target geneexpressed exactly.(4)3high expression virus strain was obtained by screening and purificationrecombinant virus.(5) The result of electron microscope observsion Bombyx mori hemolymph afterultracentrifugation showed advanced conformation empty capsid protein.【Conclusion】(1) obtained rabbit source polyclonal antibody which used for eukaryonexpression detection.(2)Constructed and screened baculovirus strains that high expressed HEVempty capsid protein...