CTCF_pEGFP-N1过表达载体及CTCF-shRNA真核表达载体的构建及鉴定

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1、CTCF/pEGFP-Nl过表达载体及CTCF-shRNA真核表达载体的构建及鉴定刘秋英，谢晓砚，魏玲，李冉，沈文燕，俞小琴，杨文理，覃扬(四川大学华西皋础医学与法医学院生物化学与分子生物学教研室，成都610041)摘耍：冃的：构建CTCF/pEGFP-Nl过表达载体及CTCF-shRNA真核表达载体并鉴定，为后续研究所用。方法：(1)将编码CTCF全长的pGEM-7Zf(-)/CTCF质粒经EcoRT酶切后回收并纯化CTCFDNA片段，再亚克隆到经EcoRl线性化的pEGFP-N1载体并转化到DH5a感受态宿主菌，挑取阳性克隆进行酶切鉴定。(2)根据人CTCFmRNA序列设

2、计并体外合成CTCF-shRNA寡核营酸片段，经退火形成双链后，克隆到线性化的pSIREN-Shuttle,并转化到DH5a感受态宿主菌，挑取阳性克隆进行酶切及测序鉴定。结果：(1)重组质粒CTCF/pEGFP-N1的酶切结杲表明CTCF正向插入EGFP-N1载体甲-。(2)阳性克隆的酶切及测序结果表明CTCFshRNA已经疋确插入质粒载体pSIREN-Shuttle里。结论：成功构建CTCF/pEGFP-N1过表达载体及靶向人CTCF基因的shRNA真核表达载体。关键词：CTCF结合因子；载体构建；RNA干扰中图分类号：R3ConstructionandidentiHcat

3、ionofCTCF/pEGFP-N1recombinantvectorandCTCF-shRNAexpressionvectorLIUQiuying,XIEXiaoyan,WEILing,LIRan,SHENWenyan,YUXiaoqin,YANGWenli,QINYang(DepartmentofBiochemistryandMolecularBiology,WestChinaSchoolofPreclinicalandForensicMedicine,SichuanUniversity,Chengdu610041)Abstract:Purpose:Toconstruct

4、andidentifyaCTCF/pEGFP-N1recombinantvectorandaCTCF-shRNAeukaryoticexpressionvectorforfurtherstudy.Methods:(1)Thefull-lenghtcDNAencodingCTCFfragmentfromthepGEM-7Zf(-)/CTCFvectorwassubclonedintoEcoRI-digestedpEGFP-N1vectorandthentransfonnedintoDH5acompetentcell.Andtheplasmidwasidentifiedbyres

5、trictionanalysis.(2)ApairofshortchainoligonucleotidestargetedtohumanCTCFmRNAweredesignedandsynthesizedbasedonthesequenceofhumanCTCFmRNA,andthenannealedtoformDNAduplex・TheDNAsegmentwasclonedintopSIREN-ShuttlevectorandtherecombinantplasmidwassubsequentlytransformedintoDH5acompetentcell・Andthe

6、plasmidwasidentifiedbyrestrictionanalysisandsequencing.Results:(1)TheresultsshowedthattheCTCFDNAfragmentwasinsertedintopEGFP-N1vectorcorrectly.(2)TheresultsofrestrictionanalysisandsequencingshowedthatCTCF-shRNAexpressionduplexwassuccessfullyinsertedintothepSIREN-Shuttleplasmidvector.Conclus

7、ions:TheCTCF/pEGFP-N1recombinantvectorandCTCF-shRNAeukaryoticexpressionvectorwereconstructedsuccessfully.Keywords:CTCFbindingfactor;vectorconstruction;RNAinterference0引言CTCF结合因子是一个广泛存在于真核生物中、具有高度保守性、功能多样性的核蛋白某金项hh高等学校博士学科点专项科研基金(20100181110051)；国家自然科学某金(