【摘要】 目的检测北京/W系和非北京/W系结核分枝杆菌HspX、Hsp65、38kDa、Ag85B和MPT64在基因、mRNA和蛋白水平有无差异,探讨5个重要蛋白对于北京/W系菌株广泛流行的可能作用。方法收集结核分枝杆菌临床分离株,其中北京/W系与非北京/W系菌株各30株,运用DNA直接测序法检测北京/W系和非北京/W系菌株HspX、Hsp65、38kDa、Ag85B和MPT64对应编码基因的PCR扩增产物,采用实时定量PCR技术检测细菌在对数生长期5个重要蛋白的编码基因mRNA的表达水平,使用Western blot技术检测细菌感染巨噬细胞24h后5个蛋白在不同菌株中的表达水平。结果统计分析采用t检验,P<0.05为差异有统计学意义。结果 HspX、Hsp65、38kDa、Ag85B和MPT64对应编码基因的PCR扩增产物测序均未发现突变。HspX、Hsp65在北京/W系菌株中的mRNA在对数生长期的表达差异均无统计学意义(P>0.05),而在感染巨噬细胞24h后,细菌中两个热休克蛋白的表达高于非北京/W系菌株,差异有统计学意义(P<0.05)。在细菌感染细胞前后,北京/W系菌株中38kDa、Ag85B的mRNA和蛋白水平的表达均低于非北京/W系菌株,差异有统计学意义(P<0.05)。MPT64在mRNA和蛋白水平的表达差异均无统计学意义(P>0.05)。结论与非北京/W系菌株比较,北京/W系菌株中HspX、Hsp65高表达,38kDa、Ag85B表达较低,推测这几个蛋白表达的改变可能与北京/W系菌株的流行有一定的关系。更多还原

【Abstract】 In order to evaluate the potential roles of HspX,Hsp65,38kDa,Ag85B,and MPT64in Beijing/W lineage Mycobacterium tuberculosis(M.tuberculosis)in Xinjiang,we studied the differential expression of these proteins in Beijing/W lineage and non-Beijing/W lineage strains.The sputum samples were cultured and isolated and the strains were identified.The genes encoding HspX,Hsp65,38kDa,Ag85B,and MPT64were amplified by PCR from genome of clinical isolates including 30Beijing/W lineage and 30non-Beijing/W lineage strains,and then were sent to sequencing.The genes mRNA levels were determined by quantitative real-time PCR.Western-blot was used for the detection of protein levels of the five genes,and than the RAW264.7macrophage was infected with M.tuberculosis after 24hours.The results showed that the five genes of clinical isolates in Xinjiang had not mutated.Compared with non-Beijing/W lineage strains,no significant difference on the expression of HspX and Hsp65mRNA in Beijing/W lineage strains was found(P>0.05),but the 38kDa and Ag85BmRNA levels in Beijing/W lineage strains reduced significantly(P < 0.05).The levels of HspX and Hsp65proteins were higher and those of the 38kDa and Ag85Bproteins were lower in Beijing/W lineage strains than that in non-Beijing/W lineage strains after the interaction between M.tuberculosis and macrophage after 24hours(P<0.05).There was no difference in MPT64mRNA and protein levels(P>0.05).In conclusion,the changes of HspX,Hsp65,38kDa,and Ag85Bmight be associated with the widespread popularity of Beijing/W lineage M.tuberculosis.更多还原