CAJ | 학술논문

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.
목적 구건단백매R기인원핵표체계통,초보탐토단백매R( gingipain R,Rgp)여만성아주염적관계.방법 구건Rgp원핵표체계통,이중조표체적rRgp작용우인단핵세포THP-1주,용류식세포술검측세포막표면CD-14표체적변화,병용매련면역흡부측정검측세포분비백세포개소(IL)-1β수평적변화.결과 확증적Rgp기인여이보도적기인서렬진행비교,핵감산서렬화안기산서렬적동원성균＞97％.THP-1세포표체CD-14적평균형광강도위68.97,rRgpAcat혹rRgpB작용우THP-1세포0.5h후,CD-14적평균형광강도분별감소도45.30、46.47,차이유통계학의의(P＜0.05),rRgp대THP-1세포분비IL-1β적수평야유명현적조단작용(P＜0.01).결론 성공구건료Rgp기인원핵표체계통,rRgp단백매능구강해CD-14,조단THP-1세포염증인자적분비,종이가능연완염증적진정,도치염증적만성화.
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.