人颗粒溶素活性片段原核表达载体的构建与鉴定

目的构建人颗粒溶素(GNLY)活性片段原核表达载体。方法采用RT-PCR技术从健康人外周单个核细胞中扩增出颗粒溶素活性片段cDNA,将其与表达载体pGEX-4T-1经限制性核酸内切酶双酶切后连接,转化E.coliBL21,挑取阳性菌落,抽提重组质粒并鉴定。IPTG诱导表达GST融合蛋白,亲和层析纯化,SDS-PAGE分析表达和纯化结果,抑菌圈法鉴定表达产物的活性。结果重组质粒插入基因序列测定证实为颗粒溶素活性片段cDNA。表达产物经SDS-PAGE分析,在相对分子质量29000处出现一明显条带,与理论结果相符。抑菌试验表明,融合蛋白分子凝血酶切产物对革兰阳性的金黄色葡萄球菌ATCC25938有抑制作用。结论已成功构建了人颗粒溶素活性片段原核表达载体,为进一步研究其抗菌、抗肿瘤作用奠定了基础。

Construction and Characterization of Prokaryotic Expression Vector for Active Segment of Human Granulysin

Objective To construct a prokaryotic expression vector for active segment of human granulysin(GNLY).Methods Amplify the cDNA encoding human GNLY from healthy human peripheral blood mononuclear cells by RT-PCR and insert into expression vector pGEX-4T-1.Digest the constructed recombinant plasmid with restriction endonuclease,then transform to E.coli BL21 for expression under induction of IPTG.Purify the expressed fusion protein by affinity chromatography,identify by SDS-PAGE and test for activity by bacteriostasis test.Results The amplified gene fragment was proved as cDNA encoding GNLY by sequencing.SDS-PAGE showed that the relative molecular mass of expressed fusion protein was 29 000,which was identical to that in theory.The fusion protein digested with thrombin inhibited the growth of Staphylococcus aureus ATCC25938.Conclusion The prokaryotic expression vector for active segment of human GNLY was successfully constructed,which laid a foundation of study on antibacterial and anti-tumor effect of GNLY.