Abstract:

Objective To clone the glyceraldehydes 3-phosphate dehydrogenase（GAPDH） gene of Porphyromonas gingivalis（P.gingivalis） and to induce its fusion expression in E.coli. Methods GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-TGAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET -32a - GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl β-D-1-thiogalactopyranoside（IPTG） of different density. Results DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. Conclusion The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

Key words: Porphyromonas gingivalis, glyceraldehydes 3-phosphate dehydrogenase, prokaryotic expression