Construction and the Recombinant Protein Expression of Human PAK4 Gene Fusion Plasmid / 中国医科大学学报

ABSTRACT

Objective To construct the expression plasmid of human PAK4 gene and identify its recombinant protein expression.Methods Total RISA was extracted from human breast cancer MCF-7 cells.The hPAKA coding sequence was amplified by polymerase chain reaction (PCR) method and subcloned into pEBG vector.After the target region was sequenced.the plasmid was transfected into HEK293 cell line.The expression of the recombinant plasmid in HEK293 cells was proved by Western blot.Results hPAKA had been constructed into the expressing vector pEBG successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of pEBG-hPAK4 fusion protein was detected by Western blot,with a molecular weight 94 KDa and was pulled down by Qutathione Sepharose 4B.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,and the expression of pEBG-hPAK4 fusion protein was identified and pulled down by Glutathione Sepharose 4B.