人肝再生增强因子真核表达及其对顺铂诱导肝癌细胞凋亡的影响  

Eukaryotic expression of human augmenter of liver regeneration and its effects on the apoptosis of QGY cells induced by cisplatin

作　　者：郑钰[1] 李小芳[1] 孙航[1] 刘杞[1] 

机构地区：[1]重庆医科大学附属第二医院感染科、教育部感染性疾病分子生物学重点实验室,重庆400010

出　　处：《重庆医科大学学报》2015年第8期1065-1070,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30971334)

摘　　要：目的:构建人肝再生增强因子(human augmenter of liver regeneration,h ALR)15k D亚型的真核表达系统,并探索ALR对顺铂(Cisplatin,DDP)诱导的人肝癌细胞株QGY凋亡的影响。方法:用聚合酶链反应(polymerase chain reaction,PCR)方法和基因重组技术,从重组质粒p PIC9K-rh ALR中扩增出编码rh ALR基因片段,将其克隆入p PICZαA质粒中构建重组质粒p PICZα-A-rh ALR。重组质粒SacⅠ酶切线性化后电转入酵母GS115中,并在1%的甲醇诱导下表达。表达上清蛋白经Western blot(ALR多克隆抗体和His-tag标签抗体)鉴定和镍柱亲和层析纯化。MTS试剂检测rh ALR体外对人肝癌细胞(Hep G2、QGY)的促增殖活性,及流式细胞仪检测rh ALR在顺铂诱导QGY细胞凋亡中的抗凋亡作用。结果:PCR、双酶切、DNA测序均鉴定重组质粒构建与预期一致。分泌表达的rh ALR约占上清总蛋白的70%,目的分子量约17 k D,Western blot均可见单一条带。纯化后的rh ALR对Hep G2和QGY的体外促增殖作用呈浓度依赖性增强(Hep G2组:F=246.729,P=0.000;QGY组:F=246.004,P=0.000),且在QGY细胞顺铂诱导凋亡时也发挥着浓度梯度式抗凋亡作用(F=101.061,P=0.000)。结论:成功构建高效分泌表达rh ALR的p PICZα-A-rh ALR GS115真核表达系统;体外实验证实rh ALR对顺铂诱导人肝癌细胞有呈浓度依赖性的抗凋亡作用。Objective：To construct pichia pastoris GS115 expression system of 15 k D recombinant human augmenter of liver regeneration（rh ALR）,and to explore its effects on human hepatoma cell line QGY treated with cisplatin（DDP）. Methods：With polymerase chain reaction（PCR）,and gene recombination techniques,c DNA of rh ALR was obtained from recombinant plasmid p PIC9K-rh ALR and inserted into plasmid p PICZαA. The recombinant plasmid p PICZαA-rh ALR demonstrated by sequencing was linearized by digestion with SacⅠ,and transformed into GS115 with electroporation. The rh ALR expression was secreted by GS115 under the induction of 10 m L/L methanol,and purified through Nickel column affinity chromatography after it was analyzed by Western blot（ALR polyclonal antibody and His-tag mouse monoclonal antibody）. The effects of rh ALR on in vitro proliferation of QGY and Hep G2 cells,as well as its effects on reducing cellular proliferation inhibiton rate induced by DDP,were evaluated by MTS reagent. The anti-apoptosis effects of rh ALR on QGY cells apoptosis induced by DDP were detected by flow cytometry. Results：The recombinant plasmid p PICZα-A-rh ALR was identified by PCR,restriction enzyme reaction methods and direct sequencing respectively. Rh ALR as a secretory protein was successfully expressed by GS115;with molecular weight about 17 k D,it accounts for 70% of the total protein in the supernatant from p PICZαA-rh ALR GS115. The results of Western blot all showed the specific single band. The high qualitative rh ALR purified through Nickel column affinity chromatography could stimulate in vitro proliferation of human hepatoma cell lines（Hep G2 and QGY）in a dose-dependent manner（F=246.729,P=0.000;F=246.004,P=0.000）,reduce cellular proliferation inhibition rate and play anti-apoptosis effect also in a dose-dependent manner when QGY cells treated with DDP（F=101.061,P=0.000）. Conclusion：The rh ALR as a secretory protein is expressed in GS115 efficiently and purified through Nickel column aff

关 键 词：肝再生增强因子 酵母表达 生物学活性 镍柱亲和层析 

分 类 号：R575[医药卫生—消化系统]