Objective To construct anti-miR-221 RNA interference lentiviral vector and screen an effective target sequence in doder to providing a new gene approach of giloma. Methods The double-stranded DNA oligo sequence including the interferential sequence was synthesized and directly connected onto the enzyme-digested vector. The product was subsequently converted into the competent cells. Grown Clone was identified by PCR. The positive clones were the builded RNA interference vector with a successful gene. The target gene and vector were digested by two types of enzyme , then purified and directionally connected. The connected products were then translated into bacterial competent cells. The positive clones identified by PCR were sequenced, analyzed and compared to obtain the vectors able to express fusion protein. Two types of plasmid were then cotransfected into 293T cells, and were detected for effective target sequence via western bolt. Results Recombinant plasmid was sequenced to confirm that the transcription template was inserted into the appropriate plasmid completely and properly. The target gene named PscSI576 was found to be interfered after co-transf'ection. Conclusion This experiment was successfully constructed the human miR-221 gene RNA interference lentiviral vector, and one effective with the highest inhibition eff'iciency was screened out.%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DNA oligo 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western bolt 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.
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