针对miR-221基因干扰慢病毒载体的构建及其有效靶序列的筛选与检测

目的：构建携带人miR-221基因的miRNA干扰慢病毒载体并寻找其有效靶序列,为胶质瘤的研究提供一种新的方法。方法：合成含干扰序列的双链DNAoligo直接连入酶切后的RNA干扰载体上。将产物转入细菌感受态细胞,对长出的克隆进行PCR鉴定,阳性克隆即为目的基因RNA干扰慢病毒载体质粒。再将目的基因与目的载体分别进行双酶切,纯化酶切产物后进行定向连接,其产物转入细菌感受态细胞,再对PCR鉴定阳性的克隆进行测序和分析比对,比对正确即为融合蛋白过表达质粒载体,然后将两种质粒共转染入293T细胞,用westernbolt法检测其有效敲减靶序列。结果：重组质粒经测序鉴定证明各转录模板完整、正确插入到相应质粒中,共转染后发现编号为PscSI576的靶点干扰效果最好。结论：本实验成功构建了人miR-221基因的RNA干扰慢病毒载体,并找到了有效的干扰靶序列。

Construction of miRNA Interference Lentiviral Vector with Human miR-221 Gene

Objective： To construct anti-miR-221 RNA interference lentiviral vector and seek the effective target sequence, to provide new gene approach for gilma investigation. Methods： To synthetic double-stranded DNA oligo sequence, and it was inserted into the digested vector of RNA interference directly. Then the vector was transplanted into the competent cells Bacterial competent cells. The clone was detected by PCR identification. The positive clones were the purpose of building RNA interference lentiviral vector with a successful gene. The target gene and vector were digested separately. Purified enzyme products were connected or re-orientationed later. The products were translated into bacterial competent cells. The positive clones of PCR were sequenced and analysis and comparison. The right clone will be the success of fusion protein expression vector. Two plasmids were then co-transfected into 293T cells, and effective target sequence was detected by western bolt. Results： Recombinant plasmid was inserted into the plasmid successfully and completly,,the target number PscSI576 was found being the best target for interference after coransfection. Conclusion： The human miR-221 gene RNA interference lentiviral vector was successfully constructed , and an effective target sequence was found.