志贺毒素2A-促黄体生成素释放激素重组毒素的构建、表达及其意义

Evaluation of construction and expression shiga toxin2A-luteinizing hormone releasing hormone recombinant toxin

目的 构建一种对子宫颈癌HeLa细胞有特异性杀伤作用的志贺毒素 2A 促黄体生成素释放激素 (Stx2A LHRH)重组毒素 ,以探索其成为新型靶向药物的可能性。方法 用聚合酶链反应(PCR)技术扩增Stx2A LHRHDNA片段 ,克隆至pET 2 0b(+)质粒中 ,酶切鉴定及测序证明成功地构建了重组毒素表达质粒pET Stx2A LHRH ,将其转染大肠杆菌BL2 1(DE3 )菌株后 ,以异丙基 β D 硫代半乳糖苷诱导 ,十二烷基磺酸钠 聚丙烯酰胺凝胶 (SDS PAGE)电泳及凝胶薄层扫描分析Stx2A LHRH重组毒素的表达情况。倒置显微镜下观察Stx2A LHRH对HeLa细胞的作用。结果 SDS PAGE电泳分析显示 ,在实验上清液中 ,诱导出相对分子质量为 3 80 0 0的融合蛋白 ,说明该载体表达了Stx2A LHRH重组毒素 ,且其表达量占上清液中总蛋白的 10 3 2 %。加入Stx2A LHRH重组毒素后 ,宫颈癌HeLa细胞几乎全部死亡。结论 成功构建了Stx2A LHRH重组毒素 。

Objective The purpose is to construct shiga toxin2A luteinizing hormone releasing hormone (Stx2A LHRH) recombinant toxin which can kill cancer cells specifically and try to get a new target binding drug Methods The fragment of Stx2A LHRH DNA amplified by polymerase chain reaction (PCR) were cloned into plasmid pET 20b(+) vector The recombinant plasmid pET Stx2A LHRH was contructed successfully and identified by endonuleases digestion and sequencing analysis then it was transformed to Escherichia coli BL21(DE3) and expressed under the induction of isopropyl β D thiogalac topyranoside Results The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel thin layer scanning proved a special band of a molecular weight of about 38 000 , accounting for 10 32% of total amount of the supernatant protein Stx2A LHRH recombinant toxin can kill HeLa cells clearly Conclusion Stx2A LHRH recombinant toxin may become a choice of target binding drug in the future.