摘要
目的构建人细胞色素P450 2C9(CYP2C9)及突变体CYP2C9*8与CYP2C9*27的慢病毒表达质粒,并于HEK293T细胞中稳定表达。方法反转录人肝总RNA获得c DNA,PCR扩增获得CYP2C9编码序列,并克隆至载体MigR1上,获得慢病毒表达重组质粒MigR1-CYP2C9。以该质粒为模板,重叠PCR方法分别引入449G>A与449G>T点突变,并分别克隆至MigR1,获得MigR1-CYP2C9*8与MigR1-CYP2C9*27慢病毒表达质粒。在HEK 293T细胞中进行病毒包装,并采用获得的慢病毒感染HEK293细胞,经流式分选和单克隆挑取获得稳定表达CYP2C9、CYP2C9*8与CYP2C9*27的细胞,命名为293T-2C9、293T-2C9*8与293T-2C9*27。实时定量PCR(qRT-PCR)与Western blot检测胞内CYP2C9表达。采用CYP2C9的特异性底物双氯芬酸对CYP2C9的活性进行评价。结果成功构建MigR1-CYP2C9、MigR1-CYP2C9*8与MigR1-CYP2C9*27表达载体。采用构建病毒感染获得的单克隆细胞293T-2C9、293T-2C9*8与293T-2C9*27在30代后荧光显微镜下观察可见绿色荧光表达率在100%,qRT-PCR与Western blot均表明有目标分子表达。提取自这三株细胞的微粒体均可催化双氯芬酸代谢为4-羟基双氯芬酸。结论成功构建稳定表达CYP2C9、CYP2C9*8与CYP2C9*27人源化细胞模型,该方法构建的细胞可用于CYP2C9*8与*27两个突变体的功能研究。
Objective To construct lentiviral expression plasmids of CYP2C9,CYP2C9*8 and CYP2C9*27,and to express CYP2C9,CYP2C9*8 and CYP2C9*27 stably in HEK2C9T cells.Methods CYP2C9 coding region was obtained by reverse transcription PCR from the human liver total RNA,and then cloned into the mammalian expression vector MigR1.CYP2C9*8 or CYP2C9*27 coding sequences were obtained from MigR1-CYP2C9 by overlapping PCR and then cloned into MigR1.The coding sequences were introduced to HEK293 cells by lentivirus,and 293T-2C9,293T-2C9*8 and 293T-2C9*27 were screened by flow cytometry and monoclonal picking.Cellular fluorescence,qRT-PCR,Western blot and the metabolic activity assays were used to identify the cells.Results MigR1-CYP2C9,MigR1-CYP2C9*8 and MigR1-CYP2C9*2293T-2C9*8 plasmids were constructed.All monoclonal cells 293T-2C9,293T-2C9*8 and 293T-2C9*27 cell lines expressed GFP after 30 passages.Both qRT-PCR and Western blot assay showed CYP2C9 expression in the cells.Microsomes of the three cell lines were capable of catalyzing diclofenac to 4-OH diclofenac.Conclusion 293T-2C9,293T-2C9*8 and 293T-2C9*27 cell lines stably expresse CYP2C9,CYP2C9*8 and CYP2C9*27,respectively.The cell lines provide useful tools for the function investigation of CYP2C9*8 and CYP2C9*27.
作者
廖凯
刘勇
万子衿
李巍
LIAO Kai;LIU Yong;WAN Zijin;LI Wei(Medical College of Yangzhou University,Yangzhou,Jiangsu,225001;Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Geriatric Diseases in Jiangsu,Yangzhou,Jiangsu,225001;School of Life and Medicine of Dalian University of Technology,Panjin,Liaoning,124221)
出处
《实用临床医药杂志》
CAS
2018年第7期1-6,共6页
Journal of Clinical Medicine in Practice
基金
江苏省高校教育厅自然基金项目(14KJB310026)
江苏省扬州市自然科学基金(YZ2014020)
扬州大学创新培育项目
