目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pG...目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pGL01载体上;使用大肠埃希菌BL21(DE3)进行PPE68片段蛋白的原核表达,在IPTG(isopropylβ-D-thiogalactoside)诱导表达后,利用SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)电泳分析表达产物并使用镍离子亲和层析及凝胶过滤层析的方法对表达产物进行体外纯化。后通过胰蛋白酶切实验、质谱分析确定PPE68最稳定片段。结果生物信息学分析初步确定了PPE68可能的稳定片段为第7-354个氨基酸并成功构建pGL01-PPE68(7-354aa)表达载体,蛋白表达纯化后获得2.4mg/mL的PPE68(7-354aa)片段蛋白但纯度较低;针对纯化的PPE68(7-354aa)蛋白较杂问题进行胰蛋白酶切和质谱分析找到稳定片段,为PPE68的第7-180个氨基酸,构建质粒后在大肠埃希菌中能稳定表达并获得2.9mg/mL的纯化蛋白,蛋白纯度可达95%以上。结论成功找到结核分枝杆菌PPE68的稳定氨基酸序列,为未来PPE68蛋白晶体结构解析以及进一步结核病诊断和疫苗开发奠定基础。展开更多
Mycobacterium is a genus of bacteria with over a hundred non-pathogenic and pathogenic species, best recognized for certain members known to cause diseases such as tuberculosis and leprosy. Two novel protein families ...Mycobacterium is a genus of bacteria with over a hundred non-pathogenic and pathogenic species, best recognized for certain members known to cause diseases such as tuberculosis and leprosy. Two novel protein families important in the pathogenesis of Mycobacterium species are the PE and PPE families. These two protein families affect the antigenic profiles, disturbing host immunity. To better understand the origin and evolution of these gene families and the differences in their composition between pathogenic and non-pathogenic strains, several bioinformatic analyses were conducted both among Mycobacterium and closely related species that contain PE35 and PPE68 gene homologs. The methods included protein homology searches (BLASTP), horizontal gene transfer analysis (IslandViewer), phylogenetic analysis, gene cluster analysis and structural and functional constraints. Results revealed that PE and PPE gene homologs were not only limited to Mycobacterium, but also existed in three other non-mycobacterial genera, Rhodococcus, Tsukamurella and Segniliparus, and were possibly initially acquired from non-mycobacterial microorganisms by multiple horizontal gene transfers. Results also demonstrated that PE and PPE genes were more diverse and more rapidly evolving in pathogenic Mycobacterium as compared with non-pathogenic Mycobacterium and other non-mycobacterial species. These findings possibly shed light on the diverse functions and origins of the PE/PPE proteins among these organisms.展开更多
文摘目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pGL01载体上;使用大肠埃希菌BL21(DE3)进行PPE68片段蛋白的原核表达,在IPTG(isopropylβ-D-thiogalactoside)诱导表达后,利用SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)电泳分析表达产物并使用镍离子亲和层析及凝胶过滤层析的方法对表达产物进行体外纯化。后通过胰蛋白酶切实验、质谱分析确定PPE68最稳定片段。结果生物信息学分析初步确定了PPE68可能的稳定片段为第7-354个氨基酸并成功构建pGL01-PPE68(7-354aa)表达载体,蛋白表达纯化后获得2.4mg/mL的PPE68(7-354aa)片段蛋白但纯度较低;针对纯化的PPE68(7-354aa)蛋白较杂问题进行胰蛋白酶切和质谱分析找到稳定片段,为PPE68的第7-180个氨基酸,构建质粒后在大肠埃希菌中能稳定表达并获得2.9mg/mL的纯化蛋白,蛋白纯度可达95%以上。结论成功找到结核分枝杆菌PPE68的稳定氨基酸序列,为未来PPE68蛋白晶体结构解析以及进一步结核病诊断和疫苗开发奠定基础。
文摘Mycobacterium is a genus of bacteria with over a hundred non-pathogenic and pathogenic species, best recognized for certain members known to cause diseases such as tuberculosis and leprosy. Two novel protein families important in the pathogenesis of Mycobacterium species are the PE and PPE families. These two protein families affect the antigenic profiles, disturbing host immunity. To better understand the origin and evolution of these gene families and the differences in their composition between pathogenic and non-pathogenic strains, several bioinformatic analyses were conducted both among Mycobacterium and closely related species that contain PE35 and PPE68 gene homologs. The methods included protein homology searches (BLASTP), horizontal gene transfer analysis (IslandViewer), phylogenetic analysis, gene cluster analysis and structural and functional constraints. Results revealed that PE and PPE gene homologs were not only limited to Mycobacterium, but also existed in three other non-mycobacterial genera, Rhodococcus, Tsukamurella and Segniliparus, and were possibly initially acquired from non-mycobacterial microorganisms by multiple horizontal gene transfers. Results also demonstrated that PE and PPE genes were more diverse and more rapidly evolving in pathogenic Mycobacterium as compared with non-pathogenic Mycobacterium and other non-mycobacterial species. These findings possibly shed light on the diverse functions and origins of the PE/PPE proteins among these organisms.
