胞内分枝杆菌HBHA基因的克隆、分析及原核表达

Cloning,analysis and prokaryotic expression of Mycobacterium intracellulare HBHA gene

【目的】克隆胞内分枝杆菌肝素结合血凝素(HBHA)基因,并对其进行生物信息学分析和原核表达。【方法】应用巢式PCR克隆胞内分枝杆菌HBHA基因,利用在线分析软件对其基因序列及编码蛋白序列进行生物信息学分析。构建原核表达载体pET32a-HBHA,并进行诱导表达。【结果】胞内分枝杆菌HBHA基因完整的ORF全长618bp,编码205个氨基酸,该基因氨基酸序列与M.avium ATCC 25291有较高的相似性。HBHA蛋白为碱性、亲水性蛋白质,含12个潜在的磷酸化位点,存在抗原表位,亚细胞定位主要存在于细胞核,蛋白空间结构以α-螺旋和无规则卷曲为主。构建了pET32a-HBHA原核表达载体,其可在原核表达系统中诱导表达大小约为38ku的HBHA重组蛋白。【结论】胞内分枝杆菌HBHA是一种抗原指数较高的碱性、亲水性蛋白,可在原核表达系统中被表达。

【Objective】The study cloned the Mycobacterium intracellulare HBHA gene and conducted biological analysis and prokaryotic expression.【Method】The Mycobacterium intracellulare HBHA 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 pET32a-HBHA was constructed and expression in E.coli BL21（DE3） pLysS was inducted.【Result】The cloned ORF of Mycobacterium intracellulare HBHA gene consisted of 618 nucleotides,encoding 205 amino acids.The amino acid sequence was highly similar to Mycobacterium intracellulare and avium ATCC 25291.Mycobacterium intracellulare HBHA was a basic hydrophilic protein,which comprised 12 potential phosphorylation sites.The antigenicity was observed.HBHA mainly distributed in the nucleus and the main structures included α-helix and random coil.Prokaryotic expression vector pET32a-HBHA was successfully constructed,which could induced the 38 ku recombinant protein of HBHA in the prokaryotic expression system.【Conclusion】The Mycobacterium intracellulare HBHA was a basic and hydrophilic protein with antigenicity.It was well expressed in prokaryotic expression system.