重组人促红细胞生成素四环素调控真核表达载体的构建与鉴定

Construction and Identification of pTRE2hyg-rhEPO Tetracycline Response Eukaryotic Expression Vector

目的构建并鉴定含有四环素调控的pTRE顺式作用元件的质粒型载体pTRE2hyg-rhEPO。方法设计含BamHI和ClaI酶切位点的hEPO基因引物,采用RT-PCR法从含有rhEPO基因的CHO细胞中克隆hEPO基因片段,亚克隆至pTRE2hyg真核表达载体。转化感受态大肠杆菌Top10,酶切鉴定阳性重组子,并进行序列测定。结果经RT-PCR能扩增出604bp的片段,与预期片段大小相符,构建的重组体经酶切鉴定能切出插入片段,测序结果与预期序列完全一致。结论成功构建了重组人EPO四环素调控真核表达载体pTRE2hyg-rhEPO。

Objective To construct and identify the pTRE2hyg tetracycline response eukaryotic expression vector containing the recombinational human erythropoietin（rhEPO） gene. Methods The rhEPO gene was amplified by RT-PCR from CHO cells using the primers that introduced BamH Ⅰ and Cla Ⅰ restriction sites at its ends, subcloned into eukaryotic expression plasmid pTRE2hyg, and transformed into complement cells Top10. Positive clones were identified by restriction endonuclease digestion and sequencing. Results As expected, the DNA fragment of 604bp was amplified by RT-PCR. BamH Ⅰ and Cla Ⅰ digestion of the recombinational plasmid yielded a product of the predicted size of the insert fragment, which was confirmed by sequencing. Conclusion pTRE2hyg-rhEPO eukaryotic expression vector has been constructed successfully.