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目的克隆胞内分枝杆菌HSP 65基因,对其进行生物信息学分析与原核表达。方法应用巢式PCR克隆胞内分枝杆菌HSP 65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 ET 15b-HSP 65并进行诱导表达。结果胞内分枝杆菌HSP 65基因完整ORF基因全长1 626 bp,编码541个氨基酸。HSP 65蛋白为酸性、亲水性蛋白质,亚细胞定位主要存在于细胞质,蛋白二级结构以α-螺旋、无规则卷曲及β-折叠为主。使用同源建模法预测了HSP 65基因编码蛋白三维立体结构。试验构建p ET 15b-HSP 65原核表达载体并成功表达。结论成功克隆胞内分枝杆菌HSP 65基因,并对HSP 65蛋白进行了生物信息学分析及原核表达。
Objective To clone Mycobacterium intracellulare HSP 65 gene and analyze its expression by bioinformatics and prokaryotic method. Methods Nested PCR was used to clone the Mycobacterium intracellulare HSP 65 gene and its bioinformatics analysis was carried out by online analysis software. The prokaryotic expression vector p ET 15b-HSP 65 was constructed and induced. Results The complete ORF gene of Mycobacterium intracellulare HSP 65 gene was 1 626 bp in length, encoding 541 amino acids. HSP 65 protein is acidic, hydrophilic protein subcellular localization mainly in the cytoplasm, protein secondary structure α-helix, random curl and β-fold based. Homology modeling was used to predict the three-dimensional structure of HSP65 gene encoding protein. The prokaryotic expression vector p ET 15b-HSP 65 was constructed and successfully expressed. Conclusion The mycobacterium intracellulare HSP65 gene was successfully cloned and the HSP65 protein was analyzed by bioinformatics and prokaryotic expression.