牙龈卟啉单胞菌临床菌株fimA基因变异性和表达频率及其重组表达产物的致炎作用  

Mutation and expression frequency of fimA gene in Porphyromonas gingivalis isolates and inflammation-causing effect of recombinant product of the gene

作　　者：陈莉丽[1] 吴梦婕[1] 严杰[2] 

机构地区：[1]浙江大学医学院附属第二医院口腔科,杭州310009 [2]浙江大学医学院病原生物学教研室

出　　处：《中华微生物学和免疫学杂志》2005年第10期850-854,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金资助项目(No.399125)

摘　　要：目的了解牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)fimA基因变异性,构建fimA基因原核表达系统,鉴定其表达产物(rFimA)的致炎作用,检测FimA在Pg临床菌株中表达频率,了解FimA与慢性牙周炎的关系。方法采用高保真PCR从6株Pg临床菌株中扩增fimA基因并测定其核苷酸序列。构建fimA基因原核表达系统,制备rFimA兔抗血清。采用Western blot鉴定rFimA抗原性和免疫反应性。建立ELISA检测38株Pg临床菌株FimA表达情况和97例慢性牙周炎患者212份龈下菌斑标本中的FimA。检测rFimA诱导人脐静脉内皮EVC-304细胞分泌IL-1I、L-8、TNF-α和细胞间黏附分子-1(ICAM-1)的作用。结果6株Pg临床菌株fimA基因核苷酸序列完全相同。所构建的原核表达系统rFimA产量高达细菌总蛋白的50%左右。rFimA可与Pg全菌抗血清发生结合反应,免疫家兔可获得高效价抗体。94.7%(36/38)菌株和91.5%(194/212)龈下菌斑标本ELISA结果阳性。1和10μg的rFimA作用EVC-304细胞48 h,其分泌的IL-1α水平升高(P<0.05);但作用12 h即可出现高水平的ICAM-1(P<0.05),未发现有诱导IL-8和TNF-α的作用(P>0.05)。结论fimA基因序列保守,在Pg临床菌株中有很高的表达频率。rFimA有良好的抗原性和免疫反应性,可作为Pg血清学检测试剂盒和Pg疫苗的候选抗原。rFimA有较强的诱导细胞分泌ICAM-1的作用。Objective To find the mutation frequence of Porphyromonas gingivalis （Pg） fimA gene, and to construct the prokaryotic expression system offimA gene. To identify inflammation-causing effect of the expressed product（rFimA） and to determine the FimA expressing frequency in Pg isolates and the correlation between FimA and chronic perindontitis. Methods fimA gene from six Pg isolates were amplified by high fidelity PCR and the target amplification fragments were then sequenced. The prokaryotic expression system of fimA gene was constructed and rabbit anti-rFimA serum was prepared. Western blot was used to identify the antigenicity and immunoreactivity of rFimA. The effect of rFimA inducing EVC-304 cells to secret IL-1, IL-8, TNF-α and ICAM-1 was measured. Results The nucleotide sequences of fimA gene from the six strains were absolutely same. rFimA output of the constructed prokaryotic expression system was approximate 50% of the total bacterial proteins, rFimA could bind to antiserum against Pg and induce rFimA-immunized rabbits to produce antibody with high titer. Over 90% of the Pg isolates and 91.5 % （194/212） of the subgingival plaque samples showed positive ELISA results. The EVC-304 cells treated with 1 and 10μg rFimA for 48 h, the IL-1α level secreted by the cells were increased. No effect of rFimA inducing IL-8 and TNF-α could be found. Condttsion fimA gene has sequence conservation and high expression frequency in Pg isolates, rFimA has strong antigenicity and so which can be used as a candi- date antigen for developing serological kit and vaccine of Pg.

关 键 词：牙龈卟啉单胞菌 fimA基因 克隆表达 检测 致炎作用 

分 类 号：R780.2[医药卫生—口腔医学] R378.3[医药卫生—临床医学]