He Xiu-Yun, Zhuang Yu-Hui, Zhang Xiao-Gang, Li Guo-Li
The 309th Hospital, The Chinese People's Liberation Army, Haidian District, 100091 Beijing, China.
Microbes Infect. 2003 Aug;5(10):851-6. doi: 10.1016/s1286-4579(03)00179-5.
To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.
为了研究结核分枝杆菌H37Rv的毒力因子，利用蛋白质组学来表征有毒力的结核分枝杆菌H37Rv和减毒的结核分枝杆菌H37Ra之间蛋白质表达的差异。进行二维凝胶电泳以分离从结核分枝杆菌H37Rv和结核分枝杆菌H37Ra中提取的培养上清液蛋白。通过质谱鉴定感兴趣的蛋白斑点，然后克隆并测序编码已鉴定蛋白的基因。银染凝胶的比较显示，结核分枝杆菌H37Rv中有三个分辨率良好的蛋白斑点，而结核分枝杆菌H37Ra中没有。蛋白斑点1被鉴定为Rv2346c。蛋白斑点2被鉴定为Rv2347c、Rv1197、Rv1038c和Rv3620c，它们具有显著的同源性，并且使用胰蛋白酶消化具有相同的肽指纹图谱。没有结核分枝杆菌蛋白与蛋白斑点3匹配。结核分枝杆菌H37Rv的Rv2346c、Rv2347c、Rv1038c和Rv3620c位于结核分枝杆菌H37Ra染色体上，并且在结核分枝杆菌H37Ra的相应区域观察到多个突变。在结核分枝杆菌H37Ra中，Rv2347c和Rv3620c的第59位密码子（CAG，谷氨酰胺）被终止密码子（TAG）取代，这可能终止了多肽的延伸。这些结果证明了研究结核分枝杆菌基因产物的重要性，并表明同基因突变菌株中的细微差异可能在鉴定减毒突变中起重要作用。
