以杆状病毒为载体表达可溶性55kD人肿瘤坏死因子受体

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

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５３０ｂｐ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ＡｃＮＰＶ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（Ｌｉｇａｎｄｂｌｏｔ）分析表明表达产物分子量约在２０～２５ｋＤ之间，有三条带。

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 20～25kD.