The studies of regulation and post-translational modification of heppatitis B virus surface antigen genes expressed in the baculovirus expression vector system

• Main Authors: Tien,Nai-Yueh, 田乃月
• Other Authors: Kou,Guang-Hsiung;Lo,Chu-Fang
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/50238591364787721420

博士 === 國立臺灣大學 === 動物學系 === 82 === A series of AcMNPV transfer vectors (pAcBSdHHd vectors),under the control of the polyhedrin promoter for expression of forgein genes, surface and pre-surface proteins genes, was constructed and utilized to express in insect Sf 21-AE cells. In the present studies,two kinds of parental viral DNA includings upercoiled wtAcMNPVDNA and BaculoGoldTM linearized DNA were used Comparison of their combination efficiency was made using limitingdilution and DNA dot-blot hybridization. The result revealedthat the re- combination efficiency of linearized viral DNA washigher than that ofsupercoiled viral DNA. The level offoreign gene products of apurified double- cross over recombinant baculovirus was higher than that of unpurifiedor single-crossover recombinant baculo- viruses.The results revealed that SHBs andMHBs were glycosylated with N- linked high-mannose chain.The 22-nm HBs subviral particles were formed in insectcells which were similar in morphologicaland antigen iccharacteristics to those of 22-nm particles isolated from the plasma of chronic active hepatitis patients. A series of recombinant baculoviruses containing HBV Sor preS2-S genes, whose 5''-untranslated leader sequences of polyhedrin mRNA or the sequences near around the translation initiation site of foreign genes were different from each other,were constructed in order to assessthe influence of the leader sequence on gene expression. Comparison of the levels of HBs protein sexpressedby different recombinant baculoviruses was made using quantitative analysis of enzyme-immunoassay and Northern dot- blotanalysis of total cyto- plasmic RNA. The results suggested that the nucleotide sequences related to the 5''-untranslated leadersequence of polyhedrin mRNA were important for the HBs protein expression and the HBs syn- thesis in insect cellsdriven by the polyhedrin promoter did not appear to follow the Kozak rule.