Cloning and expression of HSP27 gene / 第三军医大学学报

ABSTRACT

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.