Construction of the prokaryotic expression system for the prtC gene of Porphyromonas gingivalis and analysis of the relationship between thelocal level of PrtC protein and the periodontal damage
10.3969/j.issn.1002-2694.2005.05.001
- VernacularTitle:牙龈卟啉单胞菌prtC基因原核表达系统构建及牙周损伤与局部PrtC水平的关系
- Author:
Ping RUAN
;
Yaodong LUO
;
Jie YAN
- Publication Type:Journal Article
- Keywords:
Porphyromonas gingivalis;
Collagenase;
prtC gene;
Cloning / expression;
Antigenicity / immunoreactivity;
PrtC / Periodontal damage
- From:
Chinese Journal of Zoonoses
2005;(5):369-374
- CountryChina
- Language:Chinese
-
Abstract:
Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.
