蛋白激酶C受体1过表达载体、干扰载体的构建及鉴定

摘要

Objective To construct and identify the over-expression and interference eukaryotic expression vectors of receptor for activated C-kinase 1 (RACK1).Methods The RACK1 gene full-length reading frame was amplified from the human hepatoma cell line Huh-7.5.1 by RT-PCR.Then we amplified the corresponding CDS by nested PCR and cloned it into PIRES2-EGFP.The monoclone was identified by PCR and DNA sequencing .At the same time, we designed and syn-thesized complementary DNA sequences of 2 pairs of short hairpin structure and a pair of negative control sequence with hu-man RACK1 gene.After annealing, they were linked into restriction enzyme digested RNAi-Ready pSIREN-RetroQ-Zs-Green vector , and then identified by enzyme digestion analysis and DNA sequencing .The constructed over-expression vec-tor and interference vectors were transfected into HUVEC cell line by liposome .Results The size of two pairs of primers amplified by PCR sequences was completely true .A total of 954 nt oligonucleotides were successfully inserted into the vec-tor PIRES2-EGFP.The inserted sequences of RNA interference vectors pRetroQ /RACK1-1, pRetroQ/RACK1-2 and pRet-roQ/HK were consistent with our expectations .The EGFP and GFP could be seen in the transfected HUVEC cell line by fluorescence microscopy .Conclusion The over-expression vector and siRNA expression vectors of RACK 1 are successful-ly constructed , and can be introduced into HUVEC cell line .%目的：构建并鉴定蛋白激酶C受体1（ RACK1）过表达及干扰真核表达载体。方法采用逆转录聚合酶链反应（ RT-PCR）从人肝癌细胞系Huh-7．5．1中扩增RACK1全长cDNA系列，用巢式PCR扩增对应的CDS，并将其克隆到PIRES2-EGFP，PCR鉴定后进行测序分析。以人RACK1基因为靶基因，设计合成两对短发夹结构的互补DNA序列和一对阴性对照序列，退火后与酶切后的载体RNAi-Ready pSIREN-RetroQ-ZsGreen进行连接，并行酶切分析和测序鉴定。将构建好的过表达载体和干扰载体用脂质体转染HUVEC细胞系。结果两对引物PCR扩增的序列大小与预期结果一致，PIRES2-EGFP/RACK1载体954个碱基成功插入到预计位点，序列完全一致。 RNA干扰载体pRetroQ/RACK1-1、pRetroQ/RACK1-2和pRetroQ/HK插入序列均与预计相符。荧光显微镜观察，转染的HUVEC细胞均表达EGFP和GFP。结论成功构建RACK1过表达载体及其RNA干扰载体，并可被成功导入HU-VEC细胞系。