Résumé
Objective:To develop an inducible system for expression of GraB in E.coli for the purpose of investion of its function on genome DNA fragment.Methods:Amplified granzyme B cDNA by RT-PCR via extracting lymphocyte total RNA.Identified with endonuclease cut and DNA sequence analysis,the gene was inserted into E.coli expression vector pTrcHis2A,the pokaryotic expression plasmid pTrcHis2A/GraB was constructed and transformed into TOP10F.Results:After 8 h of IPTG in duction, the GraB was expressed 15% of total proteins by SDS-PAGE.Western blot assay proved the expressed hGraB to be of good antigenicity and high specificity.The recombinant protein purified by affinity chromatography has effect on GraB substrate.GraB was found to work directly on the genome DNA fragments,promoting its split.Conclusion:It is found that there may exist certain novel pathway for GraB-induced apoptosis.