PSP94-TNFαD11a融合基因在NIH3T3细胞中的表达及其产物活性分析

Expression of PSP94-TNF αD11a Fusion Gene in NIH3T3 Cells and Bioassays of the Expressed Products

为了分析 PSP94- TNFαD1 1 a融合基因的表达和表达产物的生物学活性 ,将含该融合基因的质粒 pc DNA- PSP94- TNFα D1 1 a转染 NIH3T3细胞 ,72 h后收集细胞培养上清 ,并提取细胞总RNA,经 RT- PCR,得到与目的基因长度相符合的 c DNA片段 ;以 PSP94c DNA为探针 ,对 RT-PCR产物进行 Southern印迹分析 .结果表明 :转染 PSP94- TNFαD1 1 a融合基因的 NIH3T3细胞 ,其 RT- PCR产物杂交信号为阳性 .细胞培养上清用 TNF抗体行 Western印迹和 ELISA分析 ,检测结果为阳性 .生物学活性分析表明 ,细胞培养上清不仅具有 PSP94抑制人前列腺癌细胞 PC- 3生长的活性 ,而且显示出 TNFα对 L92 9细胞的细胞毒作用 .以上结果表明 ,pc DNA- PSP94- TNFαD1 1 a质粒能够正确表达目的基因 PSP94- TNFα D1 1 a,且表达的 PSP94- TNFαD1 1 a融合蛋白具有预期的双重生物学活性 .

To identify the expression and the biological activity of human PSP94 TNF αD11a,NIH3T3 cells were transfected by pcDNA\|PSP94 TNF αD11a.Total RNA from the transfected cells was analyzed by RT\|PCR.The products showed the expected length and could also hybridized with PSP94 probe.The results of Western blot and ELISA indicated that the transfected NIH 3T3 cells could express PSP94 TNF α D11a in the culture medium.Activity assay of the culture medium from the transfected NIH 3T3 showed not only suppressive effects of PSP94 on human prostate coarcinoma cell PC\|3 growth,but also cytotoxicity of TNF α on L929 cells.These results suggested that PSP94 TNF αD11a could be correctly expressed in NIH 3T3 cells,and the expressed products had the expected double effects on tumor cells.