中文摘要：

目的构建重组融合蛋白Tumstatin—TNF—α的分泌型真核表达载体并检测其在中国仓鼠卵巢细胞（CHO—K1）中的稳定表达。方法以人胚肾293细胞为材料，提取总RNA，用RT-PCR方法合成人tumstatin cDNA，将该cDNA克隆到pGEM—T载体获得重组质粒pGEM—T／tumstatin。利用PCR从pGEM—T／tumstatin和PBV220-TNF—α载体中分别扩增出sig-tumstatin—linker和linker-TNF片段，将其克隆得到sig—tumstatin—linker—TNF片段，sig—tumstatin—linker-TNF片段经酶切后，插入经同样酶切plRESneo3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—tumstatin—linker—TNF基因片段及重组载体进行验证。将重组sig—tumstatin—linker-TNF真核表达载体转染到CHO—K1对其表达状况进行检测。结果所获得的sig—tumstatin—linker—TNF片段（1．32kb）序列与报道的序列完全一致。酶切鉴定的结果表明含肿瘤抑素基因的重组plRESneo3／sig—tumstatin-linker—TNF表达载体构建成功。转染重组plRESneo3／sig—tumstatin—linker—TNF的CHO—K1表达了肿瘤抑素融合蛋白tumstatin-linker—TNF。从生长曲线结果来看，转染plRESneo3／sig—tumstatin—linker—TNF真核表达载体和转染plRESneo3的CHO—K1比未转染的CHO—K1细胞的生长速度要慢。结论成功地构建了重组人肿瘤抑素融合蛋白的真核表达载体，并获得能稳定表达人肿瘤抑素融合蛋白的CHO—K1。

英文摘要：

Objective To construct fusion protein tumstatin-TNF-α secreted eukaryotic expression vector and to detect its expression in Chinese hamster ovary（ CHO-K1 ） cells. Methods The cDNA fragment of tumstatin was obtained using a reverse transcriptase-polymerase chain reaction （RT-PCR） with total RNA extracted from 293 embryonic kidney cells. The RT-PCR product was cloned into pGEM-T vector, then the pGEM-T/tumstatin plasmid was obtained. Fragment was obtained by PCR from pGEM-T/tumstatin vector and PBV220-TNF-α vector. Colony PCR and restriction enzyme digestion were used to verify the obtained sig-tumstatin-linker-TNF fragment. The fragment was cloned into pIRESneo3 expression vector. The recombinant pIRESneo3/sig-tumstatin-linker-TNF plasmid was then transfected into CHO-K1 cells to detect the expression of sig-tumstatin-linker-TNF out of these cells. Results The obtained sig-tumstatin-linker-TNF fragment （ 1.32 kb） was identical to the sequence of reported human fusion protein tumstatin-TNF. Restriction enzyme digestion demonstrated that the recombinant pIRESneo3/sig-tumstatinlinker-TNF expression vector was successfully constructed. The transfected CHO-K1 cells successfully expressed tumstatin-linker-TNF. In addition, the CHO-K1 cells transfected with pIRESneo3 vector and the CHO-K1 cells transfected with recombinant pIRESneo3/sig-tumstatin-linker-TNF grew slower than the untransfected CHO-K1 cells. Conclusion We have successfully constructed the recombinant pIRESneo3/sig-tumstatin-linker-TNF expression vector and obtained CHO-K1 cells that can express stable tumstatin-linker-TNF.