MMP-9基因RNAi慢病毒载体的构建与鉴定

目的构建小鼠基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)RNA干扰(RNA interference,RNAi)慢病毒载体。方法针对小鼠MMP-9基因序列,利用公用网站按照RNAi序列设计原则,设计RNAi靶点序列,并合成靶序列的Oligo DNA,退火形成双链DNA,与经HpaⅠ和XhoⅠ酶切后的pGCL-GFP载体连接产生短发卡RNA慢病毒载体,用插入鉴定引物进行PCR鉴定阳性克隆并测序,应用Western blot在293T细胞中鉴定MMP-9表达的下调作用。结果PCR鉴定和DNA测序结果显示合成的含MMP-9 shRNA慢病毒载体寡核苷酸链插入正确;构建的MMP-9RNAi慢病毒载体能够抑制MMP-9的表达。结论应用基因工程技术,成功构建了小鼠MMP-9基因RNAi慢病毒载体,为应用于在体基因治疗创造了条件。

Construction and identification of lentiviral vector of MMP-9 gene RNAi

Objective To construct lentiviral vector of matrix metalloproteI_(Na)se 9 (MMP-9) RNA interference (RNAi) of mice. Methods Towards the MMP-9 gene sequences of mice, in accordance with RNAi sequence design principles using the public Web site, RNAi target sequences were designed, then the target sequences of Oligo DNA were synthesized and annealed to double-stranded DNA, connecting with pGCL-GFP vector digested by Hpa Ⅰ and Xho Ⅰ. Short hairpin RNA lentiviral vectors were constructed. The positive clones identified by PCR and sequenced. The MMP-9 expression in the downward effect in 293T cells by Western blot was studied. Results The PCR identification and DNA sequencing showed that the lentiviral vector of MMP-9 shRNA-oligonucleotide chain was inserted correctly;and MMP-9 RNAi lentiviral vectors inhibited the expression of MMP-9. Conclusion A lentiviral vector of MMP-9 gene RNAi of mice is constructed successfully by the genetic engineering technology, and it provides a condition for gene therapy in vivo.