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目的原核表达及纯化梅毒螺旋体(Treponema palludim,Tp)热休克蛋白10(Heat shock protein 10,Hsp10)。方法利用Clustal Omega软件对Tp与其他物种的Hsp10蛋白进行多序列比对并运用生物信息学方法分析Hsp10蛋白亲水性和结构。设计特异性引物,以Tp基因组为模板,PCR扩增Hsp10基因。回收PCR产物并用EcoRI和HindIII双酶切,酶切片段与同样酶切的质粒pET28a相连,构建表达重组质粒pET28a-Hsp10,转化大肠埃希菌BL21(DE3),采用IPTG诱导重组Hsp10蛋白表达,用镍离子柱纯化目的蛋白。结果 Tp Hsp10基因编码蛋白是一个亲水性蛋白,α-螺旋、无规卷曲和β片层分别占5.7%、47.7%和45.4%,三级结构与结核分枝杆菌Hsp10(PDB:1p3h.1.C)类似,可由7个Hsp10组成一个圆形结构。PCR扩增获得的Hsp10基因全长为267bp,连接到表达载体pET28a得到重组质粒pET28a-Hsp10,编码含His标签的重组Hsp10蛋白。重组质粒转化大肠埃希菌BL21(DE3),IPTG诱导表达分子质量单位为13.7ku的重组Tp Hsp10蛋白,与理论值相符。重组蛋白经镍离子柱层析纯化,获得单一条带的Hsp10。结论成功构建重组质粒pET28a-Hsp10并表达和纯化获得Hsp10蛋白,为研究其功能奠定了基础。
Objective To prokaryotic express and purify Treponema palludim (Tp) heat shock protein 10 (Hsp10). Methods Clustal Omega software was used to compare the Hsp10 protein between Tp and other species by multiple sequence alignment and to analyze the hydrophilicity and structure of Hsp10 protein by bioinformatics method. Specific primers were designed to amplify the Hsp10 gene by PCR using the Tp genome as a template. The recombinant plasmid pET28a-Hsp10 was constructed and transformed into Escherichia coli BL21 (DE3). The expression of recombinant Hsp10 protein was induced by IPTG. The recombinant plasmid pET28a-Hsp10 was transformed into E.coli BL21 Nickel ion column purification of the target protein. Results The protein encoded by Tp Hsp10 gene was a hydrophilic protein. The α-helix, random coil and β-sheet accounted for 5.7%, 47.7% and 45.4%, respectively. The tertiary structure was similar to Mycobacterium tuberculosis Hsp10 (PDB: 1p3h.1 .C) Similarly, seven Hsp10s can form a circular structure. The total length of Hsp10 gene obtained by PCR amplification was 267 bp, and was ligated into the expression vector pET28a to obtain the recombinant plasmid pET28a-Hsp10, which contained the His-tagged recombinant Hsp10 protein. Recombinant plasmids were transformed into Escherichia coli BL21 (DE3), IPTG induced recombinant Tp Hsp10 protein with a molecular mass unit of 13.7ku, in agreement with the theoretical value. The recombinant protein was purified by nickel ion column chromatography to obtain a single band of Hsp10. Conclusion The successful construction of the recombinant plasmid pET28a-Hsp10 expression and purification of Hsp10 protein laid the foundation for the study of its function.