嗜热古菌S.solfataricus小热激蛋白基因的克隆及表达

Cloning and Expression of a Novel Small Heat Shock Protein from the Hyper-thermophile Sulfolobus solfataricus in Escherichia coli

目的:从嗜热古菌Sulfolobus solfataricus中克隆一种新的小热激蛋白SsHsp14.1的基因,并研究其表达和生物活性。方法:用PCR技术以S.solfataricus基因组为模板扩增得到目的基因序列片段,并将其克隆到pET-28a(+)中,转化到E coli BL21(DE3)中经IPTG诱导表达,纯化后对产物进行生物活性测定。结果:从菌株S.solfataricus中克隆出目的基因,该基因的编码框由375个碱基组成,编码的蛋白质由124个氨基酸组成。含该质粒的大肠杆菌经诱导表达了一个与预期理论值相符的约14kDa的蛋白,利用亲和层析和凝胶柱分离纯化了重组蛋白。试验证明纯化后的重组SsHsp14.1具有分子伴侣活性,重组蛋白在体内表达时能提高E.coli细胞的耐热性。结论:成功克隆SsHsp14.1基因并表达出蛋白,并明确了其分子伴侣活性,为该热激蛋白的研究和应用奠定基础。

Objective： To eonstruct the expression system for 14kDa of SsHSP14.1 and establish its methods for expression and purification. Chaperone activity of the sHsp was tested. Method： Target gene was amplified with PCR. The PCR product was cloned into the expression plasmid pET-28a （ ＋ ）. The recombinant plasmid was transformed into E. colt BL21 （DE3）. Recombinant SsHSP14. 1 was overexpressed by IPTG induction and purified by affinity chromatography and gel column. The purified sHsp was determined by bioassay. Result： The gene was cloned and alignment results showed that the SsHSP14.1 was composed of 375 base pairs, encoding 124 amino acids. The protein about 14kDa as expected was expressed after induction with IPTG. The purified recombinant SsHSP14.1 was proved to have chaperone activity in vitro. The overexpressed SsHSP14.1 in E. colt was also proved to enhance E. colt cells viability under heat stress. Conclusion： The gene was cloned and SsHSP14.1 was expressed sueeessfuUy. It has obvious chaperone activity, which laid a foundation of sHsp further biology research.