颗粒溶素的表达纯化及生物学活性分析

目的:采用原核表达系统对颗粒溶素(granulysin,GNLY)进行体外表达,纯化及活性鉴定.方法:以体外培养的人外周血单个核细胞(PBMC)为模板,RT-PCR扩增编码GNLY的cDNA片段,插入pMD-18-T 载体,测序正确后再亚克隆到质粒pET28a( ) 中,构建重组表达质粒pET28a( )-GNLY,转化大肠杆菌BL21(DE3) plysS,IPTG诱导表达融合蛋白,包涵体经变复性后用Ni亲和层析纯化,CFU法测定蛋白活性.结果:成功地将GNLY cDNA片段插入载体pET28a( )中,构建了表达质粒pET28a( )-GNLY.经诱导在原核表达系统中以包涵体形式高效表达了相对分子质量为9 000的融合蛋白,变复性纯化后的GNLY蛋白经CFU法检测,发现其具有细胞毒活性且细胞毒作用具有剂量依赖性.结论:本实验利用原核表达系统成功地表达了具有生物学活性的GNLY,为研究其功能、作用机制及临床应用奠定了基础.

Expression, purification and bioactivity analysis of granulysin

Objective:To express the granulysin protein in E.coli and to detect its bioactivity after purification.Methods: Using cultured human peripheral blood mononuclear cells as the template,we selectively amplified the fragment coding granulysin by RT-PCR.The fragment was then inserted into the prokaryotic expression vector pET28a(+) for transforming E.coli BL21(DE3) plysS.Fusion protein expression was induced by isopropyl-Beta-D-thiogalactopyranoside(IPTG).After renaturation,the protein was purified by affinity chromatography and its bioactivity was examined by colony-forming unit.Results: We successfully inserted granulysin cDNA fragment into vector pET28a(+) and constructed the expression plasmid pET28a(+)-GNLY.The fusion protein,with a molecular weight of 9 000,was obtained through IPTG induction.After renaturation and purification,the recombinant protein was proven to be bioactive by CFU and its cytotoxicity was in a dose-dependent manner.Conclusion: The fusion protein obtained in the present study is bioactive and can be used for further study on its function,mechanism of action and clinical application.