摘要
目的构建人restin及其核定位信号序列(nuclear localization signal,NLS)、酪蛋白激酶Ⅱ(casein kinaseⅡ,CKⅡ)作用位点缺失体基因rt1、rt2的真核表达载体并检测其在非洲绿猴肾细胞COS-7中的表达。方法应用RT-PCR方法从体外培养的人胚肾细胞HEK293的mRNA中扩增出restin及其缺失体基因的编码区,克隆至pMD18T载体并测序,然后亚克隆至真核表达载体pcDNA3.1(+)3×flag,酶切鉴定正确后以脂质体法瞬时转染COS-7细胞,Western blot检测restin、rt1、rt2的表达及其亚细胞定位情况,从而确定功能位点缺失是否影响了Restin的核定位。结果测序证实PCR扩增得到的restin、rt1、rt2基因编码区序列正确;脂质体法转染COS-7细胞后检测到预期目的蛋白的表达。截取NLS/CKII位点阻滞了Restin的细胞核定位。结论成功构建了restin及其缺失体基因的真核表达载体并使其在COS-7细胞中表达并检测到功能结构域的缺失引起蛋白Restin的核定位变化。
Objective To construct the eukaryotic expressing vectors of human restin gene and its deletants rtl ,rt2 without NLS/CK Ⅱ sites,and to investigate their expression in COS-7 cells. Methods Human restin gene coding sequences were amplified from HEK293 mRNA by RT-PCR. The PCR products of restin ,rtl and rt2 were cloned into pMD18T vectors, then sequenced and subcloned into eukaryotic expressing vector pcDNA3.1 （ ＋ ） 3 × flag. These recombined plasmids were identified and transiently transfected into COS-7 ceils subsequently with lipofectin to detect expression and subcellular localization of Restins by Western blot. Results Sequencing report showed that eukaryotic expressing vectors of restins were successfully constructed. Predicted protein was investigated in COS-7 ceils after lipofectin-mediated transfection. Conclusion The successfully constructed eukaryotic expressing vectors containing restins can express correctly in COS-γ cells while deletion of functional domain can affect nuclear localization of Restin.
出处
《山西医科大学学报》
CAS
2009年第8期673-676,共4页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(30800204)
关键词
RESTIN
NLS/CKII位点
真核表达载体
核定位
restin
NLS/CK Ⅱ sites
eukaryotic expression vector
reverse transcriptase polymerase chain reaction
nuclear localization
