小鼠Mmp-9 shRNA重组慢病毒包装、筛选及干扰效果检测

Construction and identification of RNA interference lentivirus vector of mouse matrix metalloproteinase 9 gene

目的:设计、筛选、构建并鉴定Mmp-9小干扰RNA(small interfering RNA,siRNA),并构建其慢病毒表达载体,为血管化疾病的防治提供理论依据及实验室数据。方法:针对基质金属蛋白酶9靶基因设计siRNA序列,并合成小发卡RNA(short hairpin RNA,shRNA)结构的DNA,与经AgeΙ和EcoRΙ酶切的pSIL-RFP连接、转化大肠杆菌感受态细胞,构建成携带Mmp9-shRNA慢病毒质粒载体(pSIL-RFP/MMP9-shRNA),PCR及测序鉴定后,Western blot筛选出携带最佳siRNA的慢病毒质粒载体,并利用慢病毒包装质粒(pHelper1.0和pHelper2.0)将其制备成MMP9-shRNA慢病毒,并测定病毒滴度为1×1010pfu/l。结果:PCR和DNA测序证实合成的含基质金属蛋白酶9短发卡RNA慢病毒载体寡核苷酸链正确插入pSIL-RFP载体,表明实验成功构建基质金属蛋白酶9RNA干扰慢病毒表达载体。结论:成功的构建并筛选出针对小鼠Mmp9基因沉默的特异性shRNA重组慢病毒,为血管化性疾病的防治研究提供基础。

Objective：To design and sieve small interfering RNA sequence targeting mouse matrix metalloproteinase 9 gene, and to construct recombinant lentivirus for research on disease of hemangioma. Methods： It was to design the effective sequence of small interfering RNA targeting mouse matrix metalloproteinase 9 gene and to synthesis the DNA of its short hairpin DNA （shRNA） struction. The complementary DNA sequence was synthesized and cloned into the pSIL-RFP vector, which was cut by AgeIand EcoRI. It was named Mmp9-shRNA tentiviral vector （pSIL-RFP / MM Pg- shRNA）, the resulting lentiviral vector containing matrix metalloproteinase 9 shRNA, and it was confirmed by PCR and sequencing. By western blot, the best small interfering RNA sequence was certificated and best Mmp9-shRNA. With the best Mmp9-shRNA lentiviral vector, pHelperl. 0 and pHelper2. 0, Mmp9-shRNA lentivirus was successfully produced,and the titer of virus was tested. Results：It was demonstrated that the lentivir- us vector targeting mouse Mmp9 was successfully constructed,and the best interfering RNA was perfectly selected, and Mmp9-shRNA lentivirus was produced. Conclusion：The lentivirus RNAi of Mmp9 is constructed successfully.