大鼠Uqcrfs1重组质粒的构建及其在HEK293T细胞中的表达 |
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引用本文: | 孙斯凡,古艳婷,李平,王红盼,董晞,杨靖,赵婷婷,王旭.大鼠Uqcrfs1重组质粒的构建及其在HEK293T细胞中的表达[J].中国中西医结合肾病杂志,2012,13(2):103-106. |
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作者姓名: | 孙斯凡 古艳婷 李平 王红盼 董晞 杨靖 赵婷婷 王旭 |
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作者单位: | 1. 卫生部中日友好医院临床医学研究所,北京,100029;南京中医药大学,南京,210029 2. 卫生部中日友好医院临床医学研究所,北京,100029 3. 南京中医药大学,南京,210029 |
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基金项目: | 国家自然科学基金资助项目,国家自然科学基金青年科学基金资助项目 |
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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。
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关 键 词: | Uqcrfs1 重组质粒 糖尿病肾病 |
Construction of Rat Uqcrfs1 for its Expression in HEK 293T Cell |
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Institution: | SUN Sifan,GU Yanting,LI Ping,et al China-Japan Friendship Hospital Institute of Clinical Medical Science,Beijing(100029) |
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Abstract: | 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. |
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Keywords: | Uqcrfs1 Construct plasmid Diabetic nephropathy |
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