pCMV－TNFα的构建及其在动物细胞中的表达

Construction of pCMV- TNFαand Its Expression in Ani-mal Cells

以ＸｂａⅠ和ＥｃｏＲＶ双酶消化ｐＢＴ１获得肿瘤坏死因子α（ＴＮＦα）ｃＤＮＡ基因片段，以ＸｂａⅠ和ＳｍａⅠ酶切点定向克隆入质粒载体ｐＣＭＶ４，构建了人ＴＮＦα重组质粒ｐＣＭＶ－ＴＮＦα。采用ＤＮＡ—磷酸钙共沉淀法转染ＣＯＳ７和ＮＩＨ３Ｔ３细胞，收集转染后不同时间的细胞培养上清，检测ＴＮＦα的表达水平和生物学活性。ＥＬＩＳＡ检测表明，两种细胞转染后，其培养上清中均有ＴＮＦα抗原存在，而载体等对照则测不出ＴＮＦα。以Ｌ９２９细胞检测其细胞毒活性，发现ＣＯＳ７细胞于转染后４８ｈ表达水平最高，活性高达１６Ｕ／ｍＬ以上，但７ｄ时测不出ＴＮＦα活性；ＮＩＨ３Ｔ３细胞于转染后２４ｈ活性最高（８Ｕ／ｍＬ），９ｄ时尚有表达，但不足１Ｕ／ｍＬ。

The recombinant plasmid pCMV-TNFα was constructed with plasmidvector pCMV4.And with it,COS7 and NIH3T3 cells were transfected with DNA-calci-um phosphate preparation,The exprssion level and the biological activity of TNFα inthe cell culture supernatats collected at different times were tested.By using ELISA,the TNFαantigen has been determined in the supernatats of two cell cultures,but not inthe vector control.The cytotoxicity was detected with L929 cell.The expression levelin COS7 cell at 48 h after transfection was the highest and the biological activity was upto 16 U/mL,And TNFαcould be determined at 24 h and 72 h after transfection ,butnot at 7 d.The biological activity was over 8 U/mL at 24 h transfecting NIH3T3 celland 2 U/mL at 48 h,but a few of L929 cells were destroyed at 9 d after transfection andthe biological activity was under 1 U/mL.