Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis.

PURPOSE :To clone the peptidylarginine deiminase(PAD) gene of Porphyromonas gingivalis (P.gingivalis) and to be expressed in E. coli under the best conditions. METHODS: With P.gingivalis strains ATCC33277 genomic DNA as a template, PCR was applied to obtain gene PAD which was then inserted into linear cloning vector PMD-18-T to construct clone recon. Recombinant PMD18--T-PAD was cloned and analyzed with PCR and restriction endonuclease, and PET--28a expression vector was digested by Xhol and Ncol,their products were linked to construct expression plasmid PET--28a--PAD under certain connection system. The recombinant expression plasmid PET-28a--PAD which had been confirmed correctly was transformed to E. coli competent cells BL21 and induce the expression of PAD with IPTG of different density and time. With His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot. RESULTS:DNA sequencing showed that the fragment was same as the sequence published in NCBI. Under the condition of 37°C, 0.5mmol/L IPTG, 250r/min shaking for 6 hours, the PAD could be highly expressed. CONCLUSIONS: The PAD is successfully cloned and expressed in E. coli which can be further used to study the immunity of PAD and the homologues antibody preparation. Supported by National Natural Science Foundation of China (30500560) and Natural Science Basic Research Plan of Shanxi Province(2006C242). [ABSTRACT FROM AUTHOR]