摘要
目的构建人TBC1D10C(hTBC1D10C)基因过表达慢病毒载体,并对其进行功能鉴定。方法利用PCR技术扩增hTBC1D10C基因片段,构建pLVX-hTBC1D10C-mCMVZsGreen-PGK-Puro过表达载体质粒,将构建的过表达载体质粒和系统包装质粒应用高效转染试剂盒转染至293T细胞,以包装rLV-hTBC1D10C-zpp,在HEK293细胞中检测病毒滴度。rLV-hTBC1D10C-zpp感染H9C2心肌细胞,蛋白免疫印迹法检测hTBC1D10C蛋白表达。结果 pLVX-hTBC1D10C-mCMV-ZsGreen-PGK-Puro过表达质粒构建成功,rLV-hTBC1D10C-zpp包装顺利,滴度为1×10^8 TU/ml,感染H9C2心肌细胞后,hTBC1D10C蛋白表达上调(P<0.05)。结论 rLV-hTBC1D10C-zpp构建成功,在心肌细胞中具有高效表达hTBC1D10C的效应。
Objective To construct a lentivirus vector overexpressing human TBC1D10C(hTBC1D10C) gene and identify its function. Methods The hTBC1D10C gene fragment was amplified by PCT. Human TBC1D10C gene fragment was inserted into the vector overexpressing pLVX-hTBC1D10C-mCMV-ZsGreenPGK-Puro. Then, the vector plasmid overexpressing pLVX-hTBC1D10C-mCMV-ZsGreen-PGK-Puro and system packaging plasmid were transfected into 293 T cells using HET high-efficiency transfection reagent to package rLV-hTBC1D10C-zpp. The viral titer was detected in HEK293 cells. H9C2 myocardial cells were infected with rLV-hTBC1D10C-zpp. The expression level of hTBC1D10C protein was detected by Western blot. Results The plasmid overexpressing pLVX-hTBC1D10C-mCMV-ZsGreen-PGK-Puro was successfully constructed. rLV-hTBC1D10C-zpp was successfully packaged with a titer of 1×10^8 TU/ml. The expression level of hTBC1D10C protein was significantly up-regulated after H9C2 myocardial cells were infected(P<0.05). Conclusion rLV-hTBC1D10C-zpp is successfully constructed, which can overexpress hTBC1D10C in cardiomyocytes.
作者
程阔菊
吴庆
周殿儒
冯胜红
罗健华
黄河
王毅
Cheng Kuoju;Wu Qing;Zhou Dianru;Feng Shenghong;Luo Jianhua;Huang He;Wang Yi(Department of Pharmacy Dazhou City Hospital of Integrated Traditional and Western Medicine,Dazhou 635000,China)
出处
《新医学》
2020年第1期22-26,共5页
Journal of New Medicine
基金
国家自然科学基金（81370210）
达州市重点科技计划项目（达市财建[2015]61号）
