MMP-2基因RNAi重组慢病毒的构建

Construction of RNAi lentiviral vector for MMP-2 gene

目的构建MMP-2基因RNAi重组慢病毒,以便客观研究该基因的作用。方法筛选确定的MMP-2基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与含H1启动子和绿色荧光蛋白(GFP)的pLVTHM载体连接产生LV2siMMP-2慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV2siMMP-2,pCMV2dR8.74和pMD2G3质粒共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果PCR和测序证实,成功构建MMP-2 siRNA的慢病毒载体LV2siMMP-2,包装慢病毒,浓缩病毒悬液的滴度为8×1010TU/L。结论成功构建人MMP-2基因RNAi慢病毒载体,为后期研究MMP-2基因在肿瘤细胞中的作用机制和基因治疗奠定基础。

Objective To construct a lentiviral vector for MMP-2 gene RNA interference （RNAi）. Methods The effective sequence of siRNA targeting MMP-2 gene was confirmed. Both sense and antisense Ohgo DNA of the targeting sequence were designed, synthesized and cloned into the pLVTHM vector, which contained H1 promoter and green fluorescent protein （GFP）. The resulting lentiviral vector containing MMP-2 siRNA was called LV2 siMMP-2, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector LV2shMMP-2, pCMV2 dRS. 74 and pMD2G and the lentivirus was produced. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of MMP-2 （LV2siMMP-2） producing MMP-2 siRNA was constructed successfully. The titer of concentrated virus was 8 × 10^10 TU/L. Conclusion The lentiviral RNAi vector for MMP-2 was constructed successfully, which provides possibility for further investigations of tumor biology.