大鼠Uqcrfs1重组质粒的构建及其在HEK293T细胞中的表达  

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

作　　者：孙斯凡[1,2] 古艳婷[1] 李平[1] 王红盼[1] 董晞[1] 杨靖[1] 赵婷婷[1] 王旭[2] 

机构地区：[1]卫生部中日友好医院临床医学研究所,北京100029 [2]南京中医药大学,南京210029

出　　处：《中国中西医结合肾病杂志》2012年第2期103-106,共4页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：国家自然科学基金资助项目(No.30973911);国家自然科学基金青年科学基金资助项目(No.30801539)

摘　　要：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。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.

关 键 词：Uqcrfs1 重组质粒 糖尿病肾病 

分 类 号：R346[医药卫生—基础医学]