Effect of recombinant product of Porphyromonas gingivalis gingipain R in periodontitis
10.3760/cma.j.issn.1002-0098.2011.s1.006
- VernacularTitle:牙龈卟啉菌蛋白酶R重组表达产物在牙周炎中的作用
- Author:
Di-Ya ZHANG
1
;
Sheng-Lai LI
;
Lie-Ping SHENG
;
Ling-Jing HU
;
Li-Li CHEN
Author Information
1. 浙江大学医学院附属邵逸夫医院
- Keywords:
Porphyromonas gingivalis;
Antigens,CD14;
Periodontitis;
Gingipain R
- From:
Chinese Journal of Stomatology
2011;46(z1):22-26
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a prokaryotic expression system of gingipain R(Rgp) genes to identify the correlation between Porphyromonas gingivalis(Pg) gingipain R and chronic periodontitis.Methods The routing Phenol-chloroform method was applied to prepare genomic DNA of Pg ATCC33277.Oligonucleotide primers were designed to amplify the RgpAcat and RgpB genes according to the published corresponding nucleotide sequences.Prokaryotic expression system was established,and Ni-NTA affinity chroma-tography was applied to extract and purify the recombinant proteins.rRgpAcat and rRgpB were applied to human monocyte THP-1 cell strain.Flow-cytometric analyses were performed to detect the expression of CD-14 on the cell surface.Enzyme-linked immunosorbent assay( ELISA ) was used to detect the inhibition of lipopolysaccharides-induced IL-1β production from human monocytes by rRgpAcat or rRgpB.Results In comparison with the corresponding sequences from GeneBank,homologies of the nucleotide sequences of the cloned RgpAcat and RgpB genes were ＞ 97％.rRgpAcat and rRgpB output of the constructed prokaryotie expression system was approximate 50％ of the total bacterial proteins.The intensity of fluorescence of expression of CD-14 by THP-1 cells was 68.97.After rRgpAcat or rRgpB treatment with THP-1 cells for 0.5 h,it reduced to 45.30,46.47 (P ＜ 0.05 ),and the level of IL-1 β was also decreased (P ＜0.01 ).Conclusions Prokaryotic expression systems of RgpAcat and RgpB genes were successfully established in this study.rRgpAcat and rRgpB could cleave monocyte CD-14,block the THP-1 cells secreting cytokines,and as a consequence sustain chronic inflammation.
