重组融合蛋白Tumstatin-TNF-α分泌型真核表达载体的构建及其在中国仓鼠卵巢细胞中的表达  

Reconstruction of fusion protein tumstatin-TNF-α secreted eukaryotic expression vector and its stable expression in Chinese hamster ovary cells

作　　者：赵亮[1] 姚丽娟[2] 孔建新[2] 濮跃晨[2] 孙安源[2] 罗以勤[2] 张林杰[1] 

机构地区：[1]安徽医科大学免疫学教研室,合肥230032 [2]安徽医科大学附属省立医院检验科,合肥230001

出　　处：《安徽医科大学学报》2008年第4期357-361,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:30672437)

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-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pIRESneo3/sig-tumstatin-linker-TNF表达载体构建成功。转染重组pIRESneo3/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pIRESneo3/sig-tumstatin-linker-TNF真核表达载体和转染pIRESneo3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。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.

关 键 词：肿瘤坏死因子α 重组融合蛋白质类 遗传载体 真核细胞 

分 类 号：R73[医药卫生—肿瘤] R341[医药卫生—临床医学]