以杆状病毒为载体表达可溶性55kD人肿瘤坏死因子受体

从培养的HeLa细胞中提取总RNA，通过反转录PCR技术， 从该总RNA中扩增了约530 bp的shTNFR55基因的cDNA,将cDNA克隆至转移载体pAcGp67B的多角 体蛋白基因启动子的下游与转移载体构建成重组转移质粒pAcTNFR。pAcTNFR与杆状病毒AcNP V的DNA共转染昆虫细胞sf9，通过同源重组形成含有shTNFR55基因的重组病毒。经空斑分析 和DNA斑点杂交获得了纯化的重组病毒AcNPVTNFR。采用肿瘤坏死因子(TNF)敏感的L929细 胞检测表达产物的生物学活性。结果表明表达产物可以中和TNF对L929的细胞毒性。蛋白配 基印迹(Ligand blot)分析表明表达产物分子量约在20～25kD之间，有三条带。

EXPRESSION OF SOLUBLE 55 kD RECEPTOR FOR HUMAN TUMOR NECROSIS FACTOR USING BACULOVIRUS VECTOR

Total RNA was isolated from HeLa cells and about 530 bp cDNA encoding shTNFR55 was amplified with RT-PCR from the total RNA. The cDNA was cloned into the downstream of polyhedrin gene promoter in transfer vector pAcGP67B, and a recombinant transfer plasmid pAcTNFR was constructed. Insect cell sf9 was co-transferred with recombinant plasmid pAcTNFR and baculovirus AcNPV DNA. Recombinant virus AcNPV-TNFR containing shTNFR55 gene were formed by homologous recombination in insect cells. They were purified by plaque assay and were confirmed by DNA dot blot. The biological activity of expressed product was determined with culturing TNF-susceptible L929 cell line, and the result showed that the expressed product could neutralize the cytotoxicity of TNF on L929 cell line. Ligand blot analysis revealed the expressed protein three discrete bands between 20-25 kD.