Construction Of Prokaryotic Expression Systems Of Porphyromonas Gingivalis PrtC And PgmA Genes And Correlation Between Periodontal Damage And Levels Of PrtC In Local

Periodontal disease is one of the common oral diseases characterized by periodontal pocket formation and alveolar bone absorption. Porphyromonas gingivalis is well confirmed as the major causing pathogen in the active location of different periodontitis, especially of chronic periodontitis (CP). Althopugh many previous published data demonstrated that endotoxin (LPS), fimbria and collagenase are the important pathogenic factors of P. gingivalis, pathogenic mechanism of the microbe remains not to be completely understood.Collagen is a major component in periodontal tissue in which type 1 collagen particularly serves as an important supporting structure for the teeth and makes up of 80% of collagen in periodontal ligament. So collagenase activity of P. gingivalis may play an important role in tissue destruction and progression of periodontitis. In 1992, Kato et al firstly reported to determine the nucleotide and putative amino acid sequences of collagenase gene named as prtC from P. gingivalis strain ATCC 53977, which has 1002 bp in size and encodes a protein of 333 amino acid residuals with a calculated mass of approximate 38 kDa. Expression product of prtC gene was able to degrade type I collagen but did not exhibit structural similarity with eukaryotic collagenases. P. gingivalis could be divided into three major serotypes a, b and c based on serological examination and all of the three serotypes carry prtC gene. Wittstock et al. (1996, 2000) reported that PrtCantibody are present in sera of approximately 85% periodontitis patients, which indicating stronger antigenicity of PrtC. The authors also demonstrated high conservation of prtC genes from six isolates of P. gingivalis based on sequencing results with over 98% similarities.It has been generally accepted that bacterial fimbria plays an important role in its pathogenic process. P. gingivalis was found to have fimbria adhering periodontal epithelial cells. Porphyromonas gingivalis outer membrane protein A (PgmA), found by Hongo et al. in 1999, possesses the function which is related to fimbrial morphogenesis. Synthesis of P. gingivalis fimbrial protein would be blocked if pgmA gene deletion. pgmA gene is immediately upstream from fimA gene, a structural gene ecoding fimbrial protein subunit, in P. gingivalis genome DNA. This gene has 1473 bp in size and encodes an outer membrane protein with molecular weight of approximately 60 kDa.It is well known that P. gingivalis is an obligate anaerobe needing high nutrition and long growth cycle during culture. It is a trival, time consuming and low positive proccess to acquire clinical P. gingivalis strains by using routine isolation culture and phenotype identification. Since high incidence of periodontitis is present, vaccination of multivalence genetic engineering vaccine prepared with surface protein antigens of dominant periodontitis-causing bacteria was considered as a possible way to prevent and control the disease. In addition, role of P. gingivalis collagenase in the process of occurrence and development of periodontitis is little understood. Therefore, we cloned prtC and pgmA genes from P. gingivalis genome DNA, constructed expression system of the two genes, established methods to extract and purify the expressed products and determined action of PrtC on damage of periodontal tissue. The result of this study would lay a fundation for further development of immune diagnostic kit and genetic engineering vaccine of P. gingivalis.Objective To construct a prokaryotic expression system of P. gingivalis prtC and pgmA genes, and identify the immunoreactivity and antigenicity of the expressed recombinant proteins and determine correlation between periodontal damage and PrtC levels in subgingival plaque samples from chronic periodontitis patients.Method Genome DNAs of P. gingivalis strains ATCC 33277 and 47A-1 were prepared by using routine phenol-chloroform method. Entire prtC and pgmA genes from the DNAs were amplified by high fidelity PCR. The nucleotide sequences of the target DNA amplificati...