摘要
目的构建HSP70真核表达质粒以用于肾脏缺血预适应中HSP70作用及机制研究。方法用肝癌细胞系HepG2,用RT-PCR方法扩增HSP70cDNA,并利用T载体作为过渡,将HSP70基因克隆到真核表达载体pAAV-MCS中。重组质粒转染NIH3T3细胞,Western-blot法鉴定重组质粒HSP70/pAAV-MCS能在哺乳动物细胞中正确表达。结果RT-PCR方法正确地扩增出了全长HSP70基因。限制性内切酶酶切和测序结果证实HSP70基因克隆完全正确。重组质粒转染哺乳动物细胞系NIH3T3后,ECL-Western-blottimg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HSP70/pAAV-MCS,为下一步研究HSP70在肾脏缺血预适应的作用及其机制打下基础。
Objective To construct HSP70 eukaryotic expressing plasmids for studying its role and mechanism in ischemia preconditioning (IP) of kidney. Methods Full-length HSP70 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) method from cell line HepG2 of liver cancer, and then cloned into eukaryotic expressing vector pAAV-MCS by using the T/A vector as intermediary. Following the recombinant plasmids transformed into NIH3T3 cells, the expression of HSP70 protein in the host cells was verified by using ECL Western blotting. Results Full-length HSP70 gene was amplified by RT-PCR method correctly. The correct cloning of HSP70 gene in pAAV-MCS was confirmed by restrictive enzyme digestion and sequencing. The results of ECL Western blotting indicated that the target gene could express in the mammalian cell line NIH3T3. Conclusion Recombinant plasmid HSP70/pAAV-MCS has been cloned successfully, which will provide the base for investigating the role and the mechanism of HSP70 in the ischemia precondition of kidney.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第3期201-204,共4页
Immunological Journal
关键词
HSP70
缺血预适应
真核表达质粒
肾脏
HSP70
Ischemia precondition
Eukaryotic rexpressing plamids
Kidney
