大鼠Uqcrfs1重组质粒的构建及其在HEK293T细胞中的表达

目的:构建大鼠Uqcrfs1的重组质粒并检测其在人胚胎肾293T细胞中的表达。方法:应用RT-PCR方法从大鼠肾脏组织总RNA中扩增出编码Uqcrfs1的cDNA,克隆至pUM-T载体并测序,然后亚克隆至真核表达载体pcDNA3.1/V5-His,酶切鉴定及测序正确后以磷酸钙共沉淀法瞬时转染HEK 293T细胞,使用RT-PCR、Western Blot方法检测重组质粒在转录及蛋白水平的表达。结果:测序结果证实PCR扩增得到编码Uqcrfs1的cDNA序列正确;磷酸钙共沉淀法转染HEK293T细胞后,在基因转录与蛋白表达水平,结果达到预期。结论:成功构建大鼠Uqcrfs1的重组质粒,该重组质粒可在HEK293T中过表达。

Construction of Rat Uqcrfs1 for its Expression in HEK 293T Cell

Objective:Constructing rat Uqcrfs1 cDNA in eukaryotic expression vector for testifying its expression in human embryo kidney 293T cell(HEK 293T).Methods:Rat Uqcrfs1 cDNA was amplified from rat kidney total RNA by RT-PCR.The amplified DNA fragment was cloned into vector pUM-T,digested with restriction enzymes,sequenced,then sub-cloned into the eukaryotic expression vector pcDNA3.1/V5-His.After restriction enzymes digestion and sequence,the recombinant plasmid DNA was transiently transfected into HEK 293T cell with calcium phosphate.The expression of rat Uqcrfs1 in 293T cells was detected by RT-PCR and Western Blot.Results:Rat Uqcrfs1 cDNA with the eukaryotic expression vector was successfully constructed.This recombinant plasmid DNA can express in 293T cell.Conclusion:The eukaryotic expression vector containing Uqcrfs1 were constructed and it can express Uqcrfs1 in HEK 293T cell.