牙龈卟啉菌蛋白酶R重组表达产物在牙周炎中的作用

摘要

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.%目的 构建蛋白酶R基因原核表达系统,初步探讨蛋白酶R( gingipain R,Rgp)与慢性牙周炎的关系.方法 构建Rgp原核表达系统,以重组表达的rRgp作用于人单核细胞THP-1株,用流式细胞术检测细胞膜表面CD-14表达的变化,并用酶联免疫吸附测定检测细胞分泌白细胞介素(IL)-1β水平的变化.结果 扩增的Rgp基因与已报道的基因序列进行比较,核苷酸序列和氨基酸序列的同源性均＞97％.THP-1细胞表达CD-14的平均荧光强度为68.97,rRgpAcat或rRgpB作用于THP-1细胞0.5h后,CD-14的平均荧光强度分别减少到45.30、46.47,差异有统计学意义(P＜0.05),rRgp对THP-1细胞分泌IL-1β的水平也有明显的阻断作用(P＜0.01).结论 成功构建了Rgp基因原核表达系统,rRgp蛋白酶能够降解CD-14,阻断THP-1细胞炎症因子的分泌,从而可能延缓炎症的进程,导致炎症的慢性化.