重组人肿瘤抑素分泌型真核表达载体的构建及其表达  

Construction of recombinant human tumstatin secreted eukaryotic expression vector and stable expression

作　　者：赵亮[1] 罗以勤[1] 孔建新[1] 聂江玲[1] 姚丽娟[1] 董行[1] 濮跃晨[1] 马筱玲[1] 

机构地区：[1]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》2009年第3期342-347,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:30672437)

摘　　要：目的构建重组人肿瘤抑素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。方法以人胚肾293细胞为材料,提取总RNA,用RT-PCR方法合成人tumstatinc DNA,将该cDNA克隆到pGEM-T载体获得重组质粒pGEM-T/tumstatin。以pGEM-T/tumstatin为模板扩增含Ⅳ型胶原信号肽sig基因的sig-tumstatin基因片段,sig-tumstatin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-tum-statin基因片段及重组载体进行验证。将重组sig-tumstatin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片段(822bp)序列与报道的序列完全一致。酶切鉴定的结果表明含重组人肿瘤抑素的pIRESneo3/sig-tumstatin表达载体构建成功。转染重组pIRESneo3/sig-tumstatin的CHO-K1分泌表达了重组人tum-statin。另外,从生长曲线结果来看,转染了pIRESneo3/sig-tumstatin真核表达载体和空载体的CHO-K1比未转染的CHO-K1细胞的生长速度要慢一些。结论成功地构建了重组人tumstatin分泌型真核表达载体并获得能稳定表达tumstatin的CHO-K1,为开展下一步的实验奠定了基础。Objective To construct recombinant human tumstatin secreted eukaryotic expression vector and to detect its expression in Chinese hamster ovary cells. Methods The cDNA fragment of tumstatin was obtained by 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. That the type Ⅳ collagen signal peptide was taken overlapping district with tumstatin part was obtained by a polymerase chain reaction （PCR） with pGEM-T/ tumstatin. The PCR product was restriction enzyme digested, and plasmid pIRESneo3 was digested with restriction enzyme too. The sig-tumstatin fragment was cloned into pIRESneo3 expression vector. The recombinant pIRESneo3/sig-tumstatin plasmid was then transfected into CHO-K1 cells to detect the expression of sig-tumstatin out of these cells. Results The obtained sig-tumstatin fragment （822 bp） was identical to the sequence of reported human tumstatin. Restriction enzyme digestion demonstrated that the recombinant pIRESneo3/sig-tumstatin expression vector was successfully constructed. The transfected CHO-K1 cells successfully expressed tumstatin. In addition, the CHO-K1 cells transfected with pIRESneo3 vector and the CHO-K1 cells transfected with recombinant pIRESneo3/sig-tumstatin were slower than the untransfected CHO-K1 cells in the growth speed. Conclusion We have successfully constructed the recombinant pIRESneo3/sig-tumstatin expression vector and obtained CHO-K1 cells that can stable secretively express recombinant human tumstatin.

关 键 词：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]