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OALib Journal期刊
ISSN: 2333-9721
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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
DOI: 10.3724/SP.J.1145.2014.03045, PP. 775-778
Keywords: frzb,肝癌细胞hepg2,克隆,重组慢病毒,稳定细胞株
Abstract:
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（secretedfrizzledrelatedprotein）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frzbcdna片段并经测序证实；与此同时，将frzbcdna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?lentiviralexpressionsystems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，48h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westernblot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2?g/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.图4参14
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