摘要
目的 构建热休克蛋白2 7(Heatshockprotein 2 7,HSP2 7)真核表达质粒以用于肾脏缺血预适应(ischemiaPre conditioning ,IP)的作用及机制研究。方法 利用乳腺癌细胞系MCS 7,用RT PCR方法扩增HSP2 7全长基因,将HSP2 7基因克隆到真核表达载体pAAV MCS中。重组质粒转染NIH3T3细胞,Westernblot法鉴定重组质粒HSP2 7/ pAAV MCS能在哺乳动物细胞中正确表达。结果 RT PCR方法正确地扩增出全长HSP2 7基因。限制性内切酶酶切和测序结果证实HSP2 7基因克隆完全正确。重组质粒转染哺乳动物细胞系NIH3T3后,ECLWesternblot法证实了目的基因能在其中正确表达。结论 成功地克隆真核表达重组质粒HSP2 7/ pAAV MCS ,为下一步研究HSP2 7对肾脏缺血预适应的作用及其机制打下了基础。
Objective To construct HSP27 eukaryotic expression plasmids. Methods Full-length HSP27 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from breast cancer cell line MCS-7 and cloned into eukaryotic expression vector pAAV-MCS. After the recombinant plasmids transfected into NIH3T3 cells, the expression of HSP27 protein in the host cells was characterized by ECL Western blotting. Results Full-length HSP27 gene was amplified by RT-PCR correctly. The correct cloning of HSP27 gene in pAAV-MCS was confirmed by restriction enzyme digestion and sequencing. ECL Western blotting results indicated that the target gene could express in the mammalian cell line NIH3T3. Conclusion Recombinant plasmid HSP27/pAAV-MCS had been cloned successfully, which would provide the foundation for investigating the role and the mechanism of HSP27 in the ischemia precondition of kidney.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第9期844-846,共3页
Journal of Third Military Medical University
关键词
HSP27
缺血预适应
真核表达质粒
肾脏
HSP27
ischemia precondition
eukaryotic expression plamids
kidney
