携带人肝癌细胞CDK2基因的rAAV载体的构建与鉴定
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摘要
目的构建携带人肝癌细胞CDK2基因的重组腺相关病毒(rAAV)载体;方法设计能表达CDK2特异性的小干扰RNA(siRNA),构建pAKD-shRNA-CDK2重组质粒,采用磷酸钙共转染法将重组质粒pAKD-shRNACDK2、包装质粒pAAV-RC和辅助质粒pHelper共转染AAV-293细胞,采用定量PCR方法对病毒进行滴度测定;结果三质粒共转染AAV-293细胞48h后,荧光显微镜下检测到GFP绿色荧光,表明共转染成功,测定病毒平均滴度为3.56×1012v.g/ml。结论成功构建携带人肝癌细胞CDK2基因的重组腺相关病毒rAAV-shRNA-CDK2。
Objective CDK2 gene construct carrying human hepatoma cell recombinant adeno-associated virus(rAAV)vector.Methods According to human liver cancer cell CDK2 gene sequence,to design of specific shRNA sequences against CDK2.Construct recombinant plasmids pAKD-shRNA-CDK2,the recombinant plasmid pAKD-shRNACDK2,packaging plasmid pAAV-RC and helper plasmids pHelper were co-transfected AAV-293 cells using the calcium phosphate method.The titer was measured by Real-time PCR.Results Three plasmids were co-transfected AAV-293 cells after 48 h,GFP green fluorescence was detected by fluorescence microscopy,indicating the success of co-transfected;Determination of titer of packaging success rAAV-shRNA-CDK2 was 3.56×1012 v.g./ml.Conclusion The experiment was successfully constructed with the purpose of CDK2 gene recombinant adeno-associated virus rAAV-shRNACDK2.
Objective CDK2 gene construct carrying human hepatoma cell recombinant adeno-associated virus(rAAV)vector.Methods According to human liver cancer cell CDK2 gene sequence,to design of specific shRNA sequences against CDK2.Construct recombinant plasmids pAKD-shRNA-CDK2,the recombinant plasmid pAKD-shRNACDK2,packaging plasmid pAAV-RC and helper plasmids pHelper were co-transfected AAV-293 cells using the calcium phosphate method.The titer was measured by Real-time PCR.Results Three plasmids were co-transfected AAV-293 cells after 48 h,GFP green fluorescence was detected by fluorescence microscopy,indicating the success of co-transfected;Determination of titer of packaging success rAAV-shRNA-CDK2 was 3.56×1012 v.g./ml.Conclusion The experiment was successfully constructed with the purpose of CDK2 gene recombinant adeno-associated virus rAAV-shRNACDK2.
引文
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[2]Xu RA.rAAV Vector:from Virus to Clinic[M].Beijing:Science Press,2014:2-97.
[3]Zhi-Jie,Hou Xi,Luo Wei,et al.Flubendazole,FDA-approved anthelmintic,targets breast cancer stem-like ce11s[J].Oncotarget,2015,6(8):6326.
[4]Chohan TA,Qian H,Pan Y,et al.Cyclin-dependent kinase-2as a target for cancer therapy:progress in the development of CDK2inhibitors as anti-cancer agents[J].Curr Med Chem,2015,22(2):237.
[5]Asghar U,Witkiewicz AK,Turner NC,et al.The history and future of targeting Cyclin-dependent kinases in cancer therapy[J].Nat Rev Drug Discov,2015,14(2):130.
[6]Kotterman MA,Schaffer DV.Engineering adeno-associated viruses for clinical gene therapy[J].Nat Rev Genet,2014,15(7):445.
[7]Dalkara D,Sahel JA.Gene therapy for inherited retinal degenerations[J].CR Biol,2014,337(3):185.
[8]Ohmori T,Mizukami H,Ozawa K,et al.New approaches to gene and cell therapy for hemophilia[J].J Thromb Haemost,2015,13(Suppl 1):S133.
[9]Chandler RJ,LaFave MC,Varshney GK,et al.Genotoxicity in Mice Following AAV Gene Delivery:A Safety Concern for Human Gene Therapy[J].Mol Ther,2016,24(2):198.
[10]Snyder RO,Philippe M.Adeno-Associated Virus:Methods and Protocols[M].Hatfield:Humana Press,2011:4.
[11]Lisowski L,Tay SS,Alexander IE.Adeno-associated virus serotypes for gene therapeutics[J].Curr Opin Pharmacol,2015,24:59.
[12]Grieger JC,Soltys SM,Samulski RJ.Production of recombinant adeno-associated virus vectors using suspension HEK293cells and continuous harvest of vector from the culture media for GMP FIX and FLT1clinical vector[J].Mol Ther,2016,24:287.
[13]Srivastava A.In vivo tissue-tropism of adeno-associated viral vectors[J].Curr Opin Virol,2016,21:75.