应用杆状病毒载体在昆虫细胞系统中表达乙型肝炎病毒表面抗原基因

EXPRESSION OF HEPATITIS B VIRUS SURFACE ANTIGEN GENE IN INSECT CELLS USING A BACULOVIRUS VECTOR

构建了同时带有乙型肝炎病毒表面抗原基因和大肠杆菌的β-半乳糖苷酶基因的杆状病毒转移载体质粒pVL941lacHBS。应用磷酸钙沉淀技术将pVL941lacHBS DNA转入事先用AcNPV感染过的Sf9细胞,在显色剂X-gal存在下,筛选无多角体的蓝色蚀斑。经过若干次蚀斑纯化,最后获得含乙肝病毒表面抗原基因和β-半乳糖苷酶基因的重组杆状病毒R-AcV941LS。 用R-AcV941LS感染Sf9细胞,在感染后72～96小时,用RPHA和RIA分别检测组织培养上清液和细胞裂解液中的乙型肝炎病毒表面抗原含量。结果,组织培养上清液为3.83μg/1×10~6～2×10~6细胞;细胞裂解液为4.39μg/1×10~6～2×10~6细胞。乙型肝炎病毒表面抗原的合成总量为8.22μg/1×10~6～2×10~6细胞。免疫电镜观察显示表达产物呈约22nm的球形颗粒。小鼠免疫接种实验结果表明,以R-AcV941LS感染Sf9细胞表达的乙型肝炎病毒表面抗原与在哺乳动物细胞系表达的乙型肝炎病毒表面抗原具有相近似的免疫原性。

A recombinant transfer vector plasmid pVL941lacHBS carrying HBsAg gene and lac Z gene was constructed and was used to obtain recombinant AcNPV after transfection of Sf9 cells infected with AcNPV and selection of occlusion-negative dark blue plagues with the chromogenic dye X-gal.Sf9 cells infected with recombinant AcNPV ( R-AcY94lLS ) were examined for the synthesis of HBsAg by RPHA and RIA at 72-92h postinfection. Expression of HBsAg were detected both in the cell lysates and in the culture medium. The total yield was 8.22μg/l ×106-2 × 106 cells which consisted of 4.39μg/l X 106-2×106 cells in cell lysates and 3.83μg/l×106-2 × 106 cells in the culture medium. IEM showed the HBsAg produced to be aggregate of spheroid particles with a diameter of about 22-nm, being similar to those found in the plasma. Immunization test in mice revealed that the immunogenicity of HBsAg expressed in Sf9 cells infected with R-AcV94lLS was similar to those of recombinant HBsAg produced by a transformed mammalian cell line.