Cyclin G2与LDHA相互作用的鉴定分析  

Identification of the interaction between cyclin G2 and LDHA

作　　者：庄新宾 侯晓宇 李森[1] 高锦兰[1] 刘琦[1] 罗阳[1] ZHUANG Xin-bin;HOU Xiao-yu;LI Sen;GAO Jin-lan;LIU Qi;LUO Yang(Department of Medical Genomics,Key Laboratory of Medical Cell Biology,College of Life Sciences,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学生命科学学院教育部医学细胞生物学重点实验室医学基因组学教研室,辽宁沈阳110122

出　　处：《解剖科学进展》2020年第4期466-470,共5页Progress of Anatomical Sciences

基　　金：国家自然科学基金(81571440);辽宁省教育厅课题(JC2019031)。

摘　　要：目的分析细胞周期素G2(cyclin G2)与乳酸脱氢酶A(LDHA)之间的相互作用及其意义。方法将LDHA全长cDNA片段分别克隆到pGEX-5X-1和p3×FLAG-CMV-14载体上,构建pGEX-LDHA原核重组蛋白表达载体和p3×FLAG-CMV-LDHA真核重组蛋白表达载体,诱导表达并纯化GST-LDHA融合蛋白,通过GST pull-down及western blot技术分析cyclin G2与LDHA的相互作用。分别转染p3×FLAG-CMV-LDHA、pEGFP-FALGCCNG2质粒到HEK293细胞使其表达,利用带有抗FLAG蛋白标签抗体的免疫沉淀凝胶进行Co-IP实验,进一步验证cyclin G2与LDHA的相互作用。结果成功构建了pGEX-LDHA原核重组蛋白表达载体和p3×FLAG-CMVLDHA真核重组蛋白表达载体。GST pull-down实验证实了cyclin G2与LDHA在细胞外的相互作用。Co-IP实验证实了cyclin G2和LDHA在细胞内的相互作用。结论 cyclin G2与LDHA在细胞内和细胞外能发生相互作用,为研究cyclin G2生物学功能奠定实验基础。Objective To identify the interaction between cyclin G2 and LDHA. Methods LDHA gene was cloned into vector pGEX-5 X-1 and p3×FLAG-CMV-14 to construct pGEX-LDHA and p3×FLAG-CMV-LDHA respectively. Recombinant plasmids were transformed into E. coli BL21 competent cells, and fusion proteins with GST tag were expressed and purified. The interaction between cyclin G2 and LDHA were identified by GST pull-down assay. The plasmids with 3×FLAG-CMV-LDHA pEGFP-FALG-CCNG2 were transfected into HEK293 cells to reconfirm the interaction between cyclin G2 and LDHA by Co-IP assay. Results Theprotokaryotic expression vectors pGEX-LDHA and eukaryotic expression vectors 3×FLAG-CMV-LDHA were successfully constructed. The results of GST pull-down assay suggested the extracellular interaction between cyclin G2 and LDHA. The intracellular interaction between cyclin G2 and LDHA was confirmed by Co-IP assay. Conclusion cyclin G2 and LDHA can interact with each other intracellularly and extracellularly, which would lay a foundation for further study of the biological function of cyclin G2.

关 键 词：cyclin G2 LDHA 免疫共沉淀 GST pull-down 相互作用 

分 类 号：Q26[生物学—细胞生物学]