儿童结核病结核分枝杆菌临床分离株抗原基因pepA和PPE18的变异研究

Polymorphisms of pepA and PPE18 genes in clinical isolates of Mycobacterium tuberculosis in children

目的对结核抗原编码基因pepA和PPE18进行测序,了解pepA和PPE18的基因多态性。方法选取重庆医科大学附属儿童医院感染科2006年12月至2013年12月收集的结核分枝杆菌(mycobacterium tuberculosis,MTB)临床分离株168株,对应168例结核病患儿,年龄(7.8±5.7)岁,其中男孩87名,女孩81名。运用PCR方法扩增目的基因pepA和PPE18并进行测序,与结核标准株H37Rv基因序列以及从免疫表位数据库(immune epitope database,IEDB)中查询到的表位序列进行对比,分析pepA和PPE18基因序列中的变异特征。结果在168株菌株中,117(69.64%)株菌株出现了PPE18基因突变,且PPE18基因的17个T细胞抗原表位有10个发生了改变。168株临床结核分离株中,共6例(9.84%)结核菌株发生了pepA基因多态性,且未造成其T细胞抗原表位改变。结论抗原pepA基因序列较保守,PPE18存在基因高度多态性,预测会对由pepA和PPE18蛋白组成的M72疫苗的免疫功能造成影响,从而影响M72疫苗的免疫有效性。

ObjectiveTo sequence the antigen-encoding genes pepA and PPE18 from clinical isolates of Mycobacterium tuberculosis(MTB)and analyze their polymorphisms.MethodsA total of 168 clinical isolates of MTB collected from the Department of Infection of the Children’s Hospital of Chongqing Medical University from December 2006 to December 2013 were selected,corresponding to 168 cases with tuberculosis in children aged 7.8±5.7 years,with 87 males and 81 females.The target genes,pepA and PPE18,were amplified by PCR and sequenced respectively.The results of sequences were compared with the gene sequences of H37Rv,the standard strain of MTB,and the sequences of the epitopes searched from the immune epitope database(IEDB)to analyze the mutation characteristics of the gene sequences of pepA and PPE18.ResultsAmong the 168 strains,mutations in PPE18 were found in 117(69.64%)strains and 10 of the 17 T cell antigenic epitopes of the PPE18 were altered.Six(9.84%)TB strains out of 168 clinical TB isolates showed polymorphisms in pepA gene and did not result in alteration of T cell antigenic epitopes.ConclusionPepA gene sequence is conservative,and PPE18 is highly polymorphic,which is predicted to affect the immune function of the M72 vaccine composed of pepA and PPE18 proteins,thus affecting the immunization effectiveness of the M72 vaccine.