人DR5胞外区域克隆及在大肠埃希菌中表达

Cloning and expression of extracellular fragment of human DR5 in E.coli

目的克隆人死亡受体5(DR5)cDNA的胞外区域(eDR5),构建原核重组表达载体pGEX-eDR5,使eDR5在大肠埃希菌中表达。方法采用RT-PCR(反转录PCR)方法从人肝癌细胞HepG2中获得eDR5,然后克隆到pMD19-T载体上进行测序,得到正确的基因片段。经双酶切将目的基因片段插入到原核表达载体pGEX-4T-1上,测序正确后,转入大肠埃希菌BL21(DE3)中用IPTG诱导表达GST-eDR5融合蛋白,最后用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白的表达量。结果重组克隆载体pMD-eDR5经测序鉴定序列正确。构建的融合表达载体pGEX-eDR5在大肠埃希菌中表达,可溶性目的蛋白占细菌总蛋白约19%。结论成功克隆了人DR5 cDNA的胞外区域,并在大肠埃希菌中表达。为下一步分离纯化人DR5重组蛋白,制作多克隆抗体提供基础依据。

Objective To clone the extracellular fragment of human DR5 （death receptor 5 ）cDNA（eDR5） and express it in E. coli cells. Methods The eDR 5 was amplified from human liver cancer cell line HepG2 by RT- PCR （reverse tran- scription PCR）, and was cloned into the vector pMD19- T. After verified by sequencing, it was cut with restriction endonu- cleases BarnH I and EcoR I, and then the eDR5 was extracted and inserted into the prokaryotic expression vector pGEX - 4T - 1 and verified by sequencing. Finally the recombinant expression plasmid pGEX - eDR5 was transformed into E. coli BL21 （DE3）, and induced to express the GST - eDR5 fusion protein with IPTG. Expressed protein was identified by SDS - PAGE. Results Sequence analysis proved that the eDR5 was correct and prokaryotic expression plasmid pGEX - eDR5 was constructed successfully. The GST - eDR5 fusion protein was expressed in E. coli BL21 （DE3） at a high level which accounted for about 19% of the total bacterial cellular proteins. Conclusion The extraceUular fragment of human DR5 cDNA was cloned successfully and was expressed at a high level in E. coli. This research laid a foundation for purifying human DR5 polypeptides and generating anti - human DR5 polyclonal antibody.