Wnt蛋白拮抗剂Frzb的cDNA克隆、重组慢病毒构建以及稳定表达细胞株的建立  

cDNA cloning, lentiviral expression construction and stable HepG2 cell line establishment of Wnt Antagonist Frzb

作　　者：马玉玲[1] 裴哲[1] 王晶[1] 魏枭[1] 曾中秋[1] 陈雨文[1] 唐亚雄[1] 

机构地区：[1]中国科学院成都生物研究所,成都610041

出　　处：《应用与环境生物学报》2014年第5期775-778,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(81173095);四川省国际合作计划(2013HH0014)资助~~

摘　　要：Wnt信号通路的异常活化与人类多种恶性肿瘤的发生密切相关,为探索作为Wnt蛋白拮抗剂sFRP(Secreted frizzled related protein)家族成员之一的Frzb/sFRP3在肝癌细胞中的抗癌潜力,本研究主要通过RT-PCR以及慢病毒表达系统进行了Frzb的cDNA克隆、重组慢病毒构建以及肝癌稳定表达细胞株的建立.从人正常肝细胞HL-7702中提取总RNA,通过RT-PCR获得Frzb cDNA片段并经测序证实;与此同时,将Frzb cDNA分别构建至以绿色荧光蛋白或嘌呤霉素抗性为报告基因的慢病毒真核表达载体pLVX-IRES-ZsGreen1-Frzb及pLVX-Puro-Frzb,通过酶切和测序获得正确的慢病毒重组表达质粒;其次,通过Lenti-X?Lentiviral Expression Systems将慢病毒重组表达质粒与慢病毒包装质粒psPAX2、pMD2.G共转染HEK293T细胞,48 h后制备重组慢病毒悬液,随后感染HEK293T细胞,分别通过荧光显微镜观察绿色荧光蛋白的表达以及Western blot验证了Frzb(FLAG标签)在细胞中的正确表达;最后,将Frzb重组慢病毒悬液感染肝癌HepG2细胞,经2μg/mL嘌呤霉素筛选成功地获得了稳定表达Frzb的细胞株.以上结果表明慢病毒表达体系对Wnt信号通路的分子靶向药物研究具有重要价值.Aberrant activation of the Wnt signaling pathway is closely linked to tumorigenesis in a variety of major human malignant tumors. However, the anticancer potential of Frzb/sFrp3, one of the secreted frizzled related protein（sFRP） families, in hepatocellular carcinoma is poorly understood. This research aimed to study the cDNA cloning, lentiviral expression construction and stable HepG2 cell line establishment of Wnt antagonist Frzb. The Frzb cDNA fragments were amplified by reverse transcription polymerase chain reaction（RT-PCR） from the total RNA of normal human liver HL-7702（L02） cells, which were verified by the direct sequencing of PCR products. Secondly, the Frzb cDNA fragments were subcloned into the pLVX-IRES-ZsGreen1-Frzb or pLVX-Puro-Frzb, containing a fluorescent marker（GFP） or a puromycin resistance gene respectively; and the recombinant lentiviral expression plasmids were further confirmed by double digestion and sequencing. Thirdly, HEK293 T cells were co-transfected with the lentiviral expression plasmid, plus packaging plasmid psPAX2 and envelope plasmid pMD2.G by using Lenti-X？ Lentiviral Expression Systems to obtain recombinant lentiviral particles at 48 hours after transfection; then the overall titers were evaluated by fluorescence microscopy of GFP expression, and the expression of Flag-tagged Frzb successfully detected by Western blot in the infected HEK293 T cells. Finally, HepG2 cells were infected with the recombinant lentiviral particles expressing Frzb and the stable Frzb-transducted HepG2 cell line was successfully established by antibiotic selection with puromycin（2 μg/mL）. These findings help to lay a foundation for studies about anticancer activities and molecular mechanisms of Frzb in hepatocellular carcinoma.

关 键 词：FRZB 肝癌细胞HEPG2 克隆 重组慢病毒 稳定细胞株 

分 类 号：R730.2[医药卫生—肿瘤]