中国结核分支杆菌MPT64蛋白多态性及对MPT64抗原检测试剂盒的敏感性影响

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摘要目的探讨中国结核分支杆菌MPT64抗原的多态性及对其T/B细胞抗原表位的影响;应用MPT64抗原检测试剂盒对MPT64基因突变菌株和野生菌株进行检测,以进一步分析MPT64抗原多态性对结核感染检测的影响。方法从中国疾病预防控制中心传染病预防控制所结核室保存的2346株国内结核分枝杆菌复合群临床分离株中挑选180株,对MPT64基因扩增后进行序列比对,比较其T/B细胞抗原表位与非表位的多态性,对突变的菌株与野生菌株进行Bactec MGIT 960 系统与液体培养管进行培养,报阳后,用胶体金标记抗MPT64单克隆抗体采用免疫层析色谱法进行MPT64蛋白检测。结果180株临床分离株中有15株（包括2株BCG菌株）表现有MPT64蛋白的多态性,占菌株数的8.33%,8株菌有63bp的缺失,4株菌有单位点的非同义突变,1株是单位点插入。MPT64基因的突变造成13个T细胞抗原表位（56.52%）和4个B细胞抗原表位（占80%）的改变,T/B细胞抗原表位区的dN/dS值均高于对应的非表位区。8株有63bp缺失的菌株胶体金检测阴性,对应的野生株检测阳性;5株单位点突变的菌株与对应的野生菌株胶体金检测均阳性。结论中国结核分支杆菌MPT64蛋白存在多态性,这是产生抗原改变的原因之一,这会影响抗原相关功能的改变并引起免疫逃逸。63bp缺失会造成MPT64抗原检测试剂盒的假阴性,而某些单位点突变对检测效率无影响。

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	仇艳
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	刘海灿
	李马超
	万康林
	刘志广

关键词 ：结核分支杆菌, MPT64, 多态性, 胶体金

Abstract：ObjectiveTo investigate the polymorphism of the antigen MPT64 of Mycobacterium tuberculosis complex (MTBC) in China and its impact on T/B cell epitopes and to further explore its impact on detection of tuberculosis (TB) infection by an immunochromatographic assay using Mycobacterium tuberculosis( M.tuberculosis) diagnostic kit (colloidal gold method).MethodsWe selected 180 clinical isolates from 2,346 MTBC preserved in the Tuberculosis Laboratory, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Comparative sequence analysis was performed on the MPT64 genes after amplification and comparison was made between the polymorphisms of T/B cell epitopes and of non-epitopes. Then the strains with mutations and those without mutations were incubated by BACTEC MGIT 960 system. After the strains were detected positive by the system , protein MPT64 was determined by an immunochromatographic assay using anti-MPT64 monoclonal antibodies marked with colloidal gold.ResultsAmong the 180 isolates in this study, fifteen strains (8.33%, including 2 BCG strains) presented polymorphisms in the MPT64 protein. Eight of the 13 strains had a 63bp deletion, while four strains showed single-base nonsynonymous mutations and one strain had a single-base insertion . The mutations of MPT64 gene led to changes in 13 T cell epitopes (56.52%) and 4 B cell epitopes（80%）, and the values of dN/dS in the antigen epitope regions of T cell and B cell were obviously higher than those in the non-epitope regions. When being tested by M.tuberculosis diagnostic kit (colloidal gold method), eight strains with 63bp deletion showed negative results, whereas five strains with single-base mutations and all the wild strains had positive ones.ConclusionsPolymorphism of Mycobacterium tuberculosis protein MPT64 is one of the reason for its antigen change. It can lead to related functional change of the antigen and induce immunologic escape. A 63bp deletion causes false negative results in the detection of MPT64 antigen by M.tuberculosis diagnostic kit (colloidal gold method), while the single-base mutation do not affect the detection performance of the kit.

Key words： Mycobacterium tuberculosis MPT64 Polymorphism Colloidal gold method

通讯作者: 刘志广,e-mail：liuzhiguang@icdc.cn;万康林 e-mail：wankanglin@icdc.cn

作者简介: 仇艳（1986-）,安徽亳州人,汉族,硕士,主要研究方向为结核病原学及流行病学

引用本文:

仇艳,蒋毅,刘海灿,李马超,万康林,刘志广. 中国结核分支杆菌MPT64蛋白多态性及对MPT64抗原检测试剂盒的敏感性影响[J]. 实用预防医学, 2015, 22(4): 385-389. QIU Yan, JIANG Yi, LIU Hai-can, LI Ma-chao, WAN Kang-lin, LIU Zhi-guang. Polymorphism of Mycobacterium tuberculosis protein MPT64 in China and its impact on the sensitivity of MPT64-based diagnostic kit. , 2015, 22(4): 385-389.

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