重组人肿瘤坏死因子α分泌型真核表达载体的构建及表达

目的构建人肿瘤坏死因子rhTNF—α分泌型真核表达载体，并检测其在中国仓鼠卵巢细胞中的稳定表达。方法以pGEM—T／sig—tumstatin为模板，扩增与TNF带重叠区的Ⅳ型胶原信号肽sig基因片段，以PBV220-TNF为模板，扩增与sig带重叠区的TNF基因片段，以上述2个扩增产物片段为模板，重叠区扩增拼接法（SOEing）扩增得到sig—TNF。sig—TNF片段经酶切后，插入经同样酶切的pIRESneo3质粒，利用克隆PCR、限制性内切酶消化以及序列测定，对获得的sig—TNF基因片段及重组载体进行验证。将重组sig—TNF真核表达载体转染到CHO—K1，并对其表达状况进行检测。结果所获得的sig—TNF片段（558bp）序列与报道的序列完全一致，酶切鉴定结果表明含人肿瘤坏死因子的重组pIRESneo3／sig-TNF表达载体构建成功，转染重组pIRESneo3／sig—TNF的CHO—K1分泌表达了人肿瘤坏死因子rhTNF—α。转染了pIRESneo3／sig—TNF真核表达载体和空载体的CHO—K1和未转染CHO—K1细胞的生长速度没有差别。结论成功构建了重组人肿瘤坏死因子rhTNF—α分泌型真核表达载体，并获得能稳定表达rhTNF—α的CHO—K1，为开展下一步的实验奠定了基础。

Construction of Recombinant Human Tumor Necrosis Factor-alpha Secreted Eukaryotic Expression Vector and its Expression on Chinese Hamster Ovary Cells

Objective To construct human tumor necrosis factor-alpha secreted eukaryotic expression vector and to detect its stable expression in Chinese hamster ovary cells. Methods The type Ⅳ collagen signal peptide overlapping in part with TNF was obtained by a PCR with template pGEM-T/sig-tumstatin. The TNF fragment overlapping in part with sig was obtained by a PCR with template PBV220-TNF. Then the sig-TNF fragment was obtained by a splicing by overlapping extension PCR （SOEing-PCR） with the previous products. The SOEing-PCR product was digested by restriction enzyme,then inserted with plasmid pIRESneo3 which was digested by restriction enzyme too. The sig-TNF fragment and expression vector were verified by cloning PCR, restriction enzyme digestion and sequence identification. Then recombinant pIRESneo3/sig-TNF plasmid was transfeeted into CHO-K1 6ells to detect the expression of sig-TNF out of these cells. Results The transfected sig-TNF fragment （558 bp） was identical to the sequence of reported human TNF-alpha. The results of restriction enzyme digestion demonstrated that the recombinant pIRESneo3/sig-TNF expression vector was suc- cessfully constructed. The transfected CHO-K1 cells successfully expressed TNF-α. In addition, no difference in the growth speed was observed between the CHO-K1 cells transfeeted with pIRESneo3 vector,the CHO-K1 cells transfected with recombinant pIRESneo3/sig-TNF and the untransfeeted CHO-K1 cells. Conclusion It is successful to construct the recombinant pIRESneo3/sig-TNF expression vector and obtain CHO-K1 cells that can express rhTNF-α stably.