miR-148a克隆和逆转录病毒载体构建.PDFVIP

2010;30(7) 南方医科大学学报(JSouthMedUniv) 窑1545窑 miR-148a克隆及逆转录病毒载体构建 徐学虎袁全天一袁曾伟霞袁陈欣洁袁李伟明渊广州医学院第三附属医院普通外科袁广东广州 510150冤 摘要院目的 克隆微小RNAhsa-miR-148a并构建逆转录病毒表达载体遥 方法 将PCR扩增得到的miR-148a 前体序列 和pMSCV 载体经双酶切连接产生pMSCV-miR-148a 逆转录病毒表达载体袁 双酶切后测序鉴定袁 筛选阳性克隆遥 用 pMSCV-miR-148a 和PIKpackaging 质粒以磷酸钙共沉淀法转染包装细胞293FT袁 包装产生逆转录病毒遥 将病毒感染 NIH3T3 细胞进行滴度测定遥 结果 经双酶切鉴定和测序证实袁 成功构建了 miR-148a 的逆转录病毒表达载体 8 pMSCV-miR-148a遥 并测得病毒滴度为 5伊10CFU/ml遥 结论 以miR-148a 前体序列构建的逆转录病毒表达载体 pMSCV-miR-148a能够产生高滴度的逆转录病毒袁为深入研究miR-148a 的生物学功能奠定了基础遥 关键 院has-miR-148a曰DNA 甲基转移酶曰肿瘤曰甲基化曰逆转录病毒 中图分类号院Q78 文献标识码院A 文章编号院16734-254渊2010冤07-1545-03 Cloningofhsa-miR-148aandconstructionofitsretroviralexpressionvector XUXue-hu,QUANTian-yi,ZENGWei-xia,CHENXin-jie,LIWei-ming DepartmentofGeneralSurgery,ThirdAffiliatedHospitalofGuangzhouMedicalCollege,Guangzhou510150,China Abstract: Objective Toclonehsa-miR-148aandconstructitsretroviralexpressionvector. Methods Thepre-miR-148a amplifiedbyPCRwasinsertedtopMSCV toconstructtherecombinantretroviral expressionplasmidpMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148aandPIKpackagingplasmidwerecotransfectedinto293FTpackagingcellsbycalciumphosphate-mediated transfectiontoproducetheretrovirus,andtheretrovirustiterwasmeasuredbyinfectionofNIH3T3cells. ResultsRestriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed 8 successfully,andthevirustiterwas5伊10 CFU/mlafterinfectionofNIH3T3cells. ConclusionThesuccessfulconstructionof theretroviralexpressionvectorMSCV-miR-148aallowstheproductionofhigh-titerretrovirustofacilitatefurtherstudyofthe molecularfunctionsofmiR-148a. Keywords:has-miR-148a;D