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梅花鹿S100A4 基因RNAi 重组慢病毒载体的构建

魏明立 褚文辉 赵海平 王大涛 孙红梅 李春义   

  1. 中国农业科学院特产研究所
  • 出版日期:2012-11-22 发布日期:2012-11-20

Construction of a lentiviral vector to target expression of S100A4 of sika deer (Cervus nippon)

WEI Mingli,CHU Wenhui,ZHAO Haiping,WANG Datao,SUN Hongmei,LI Chunyi   

  • Online:2012-11-22 Published:2012-11-20

摘要: 本研究针对东北梅花鹿S100A4 基因筛选出若干条RNAi 靶位点,并利用NCBI 中的BLAST 工具去除脱靶情况,最终得到2 条高分靶位点。然后在T4 连接酶的作用下,将其与载体质粒pLVTHM 的酶切产物进行连接,并通过PCR 鉴定及测序筛选出阳性质粒。pLVTHM 阳性重组质粒与pMD2. G 及pCMV-dr8.91 共转染到293 T 细胞中。在倒置荧光显微镜下观察转染效果并进行收获病毒质粒。结果显示在转染24 h 后,在293 T 细胞中观察到了绿色荧光。本研究成功构建出针对梅花鹿S100A4 基因的慢病毒载体,为以后进一步研究S100A4 基因在鹿茸再生中的具体功能与机制打下了基础。

关键词: 鹿茸, S100A4, 基因, 293T, 慢病毒载体, RNAi

Abstract: In this study,a number of sequences of small interfering RNA targeting S100A4 gene of sika deer (Cervus nippon)were synthesized and screened,among which two were confirmed by BLAST search to be suitable for the purpose. Oligo DNAs containing either sense or antisense strands were ligated into the lentiviral plasmids (pLVTHM)using T4 DNA ligase. Positive clones were identified based on the results of both PCR and sequencing. Each positive plasmid was cotransfected into 293T cells with the plasmids pCMV-dr 8. 91 and pMD2. G. Twenty-four hours after the co-transfection, green fluorescence of the 293 T cells could be observed under the inverted microscope. Therefore,we successfully constructed recombinant lentiviral system targeting sika deer S100A4 gene. This work would lay the foundation for revealing the regulatory mechanism of S100A4 underlying antler generation and regeneration.

Key words: Antler, 293 T, Lentiviral vector, RNAi, S100A4 gene