CDCP1胞外段基因的克隆及在大肠杆菌中表达  

Cloning and expression of CDCP1 gene encoding its extracellular domain in Escherichia coli

作　　者：李玲霞[1] 王志巧[2] 罗晓丽[2] 宋丽娜[1] 于珊珊[1] 何成彦[1] 赵丽纯[2] 李洪军[1] 赵海丰[3] 武利涛[4] 

机构地区：[1]吉林大学中日联谊医院,吉林长春130033 [2]吉林大学药学院,吉林长春130021 [3]佳木斯大学第一临床学院,黑龙江佳木斯154003 [4]北京中医药大学东直门医院

出　　处：《中国实验诊断学》2014年第7期1063-1065,共3页Chinese Journal of Laboratory Diagnosis

基　　金：吉林省科技厅基金(2011713;20090461;20110739);黑龙江省基金(2010000742;D201075;Gc09c317);吉林省发展改革委员会(2013c026-5);长春市科技厅基金(2011125)

摘　　要：目的构建携带CDCP1胞外段基因的原核表达载体。方法以A549的mRNA为模板,应用所设计的引物通过RT-PCR法扩增CDCP1胞外段基因;将PCR产物与pGEX-KG载体连接,获得重组质粒pGEX-KG/CDCP1;经双酶切、PCR及DNA序列测定进行鉴定;IPTG诱导表达。结果①RT-PCR扩增得到约270bp大小的CDCP1胞外段基因目的片段;②目的片段正确插入到pGEX-KG中;③经IPTG诱导表达,可见在相对分子质量约35kD处出现明显的诱导蛋白条带,与预期一致。结论成功构建了携带CDCP1胞外段基因的原核表达载体,在大肠杆菌中获得大量表达。Objective To construct a prokaryotic expression vector inserted CDCP1 gene that encoding its extracel-lular domain.Methods CDCP1 target fragment was obtained by RT-PCR from A549 cell line.The recombinant pGEX-KG/CDCP1 was obtained by ligation of the PCR product and pGEX-KG.The inserted target fragment was demonstrated by digesting the recombine plasmid using two kinds of restriction enzymes,PCR and DNA sequencing.The fusion protein fusion expression was induced by IPTG.Results ①The 270 bp CDCP1 gene that encoding its extracellular domain was obtained by RT-PCR;②The target fragment was rightly inserted in the pGEX-KG;③A 35 kD induced protein was observed after IPTG treatment.Conclusion The prokaryotic expression vector inserted CDCP1 gene that encoding its extracellular domain is constructed and its fusion protein can be largely expressed in Escherichia coli.

关 键 词：CDCP1 基因克隆 融合表达 

分 类 号：R378[医药卫生—病原生物学]