胞内分枝杆菌HSP 70基因的克隆、分析及原核表达

胞内分枝杆菌为致病性非结核分枝杆菌（NTM）之一,分布广泛,人畜均可感染,对人畜健康造成极大威胁。应用巢式PCR克隆胞内分枝杆菌HSP 70基因,利用在线分析软件对其进行生物信息学分析。构建原核表达载体p ET15b-HSP 70,同时进行诱导表达。结果显示,胞内分枝杆菌HSP 70基因完整,ORF基因全长1860bp,共编码619个氨基酸,HSP 70为酸性、亲水性蛋白质,不存在明显跨膜结构,含14个潜在磷酸化位点,亚细胞定位主要存在于细胞质中,无信号肽结构。二级结构预测,蛋白空间结构以α-螺旋和无规则卷曲为主。同时,以同源建模法预测了HSP 70基因编码蛋白的三维立体结构。成功构建p ET 15b-HSP 70原核表达载体,在原核表达系统中该载体可诱导表达70ku的HSP70重组蛋白。

Cloning,Analysis and Prokaryotic Expression of HSP 70 Gene of Mycobacterium Intracellulare

Mycobacterium intracellular is a kind of pathogenic nontuberculous mycobacteria（NTM）,which is spread widely.There is a great threat to human and animal health through infection.The study which cloned the Mycobacterium intracellulare HSP 70 gene was amplified by nested PCR and the biological information analysis was performed with an online tool.At the same time,the prokaryotic expression vector p ET 15b-HSP 70 was constructed and inducted expression in E.col i BL21（DE3） p Lys S.Results showed the Mycobacterium intracellulare HSP 70 has been cloned.Its ORF consisted of 1860 nucleotides,encoding 619 amino acids.Mycobacterium intracellulare HSP 70 is an acidic and Hydrophilic protein which comprises 14 potential phosphorylation sites.Most of HSP 70 exist in the Cytoplasm.The results of secondary structural analysis showed that the main secondary structures were α-helix and random coil.At the same time,the tertiary structure was also predicted by homology modeling.In this experiment,the p ET 15b-HSP 70 as a Prokaryotic expression vector was successfully constructed,and the 70 ku recombinant protein of HSP 70 can be induced expression in the prokaryotic expression system.