抗乙型肝炎病毒C区基因的M1GS RNA核酶真核表达载体的构建
发布时间：2018-12-28 14:23
【摘要】：目的:构建特异性抗乙型肝炎病毒(HBV)C区基因的M1GS RNA核酶的真核表达载体,用于乙型肝炎的基因治疗研究。 方法:选择HBV ayw亚型C区基因2333nt为切割位点,设计M1GS RNA核酶的DNA模板的PCR引物。以含有编码M1 RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到特异性抗HBV C区基因的M1GS RNA核酶的DNA模板,并将其和真核表达载体pEGFP-C1经限制性核酸内切酶EcoR Ⅰ和Sal Ⅰ分别双酶切后回收,再行连接反应,得到重组质粒pEGFP-GS。转化大肠杆菌JM109,选取阳性克隆碱裂解法进行质粒抽提,Sal Ⅰ和EcoR Ⅰ双酶切电泳鉴定和测序鉴定。 结果:重组质粒pEGFP-GS经EcoR Ⅰ和Sal Ⅰ酶切后,酶切产物行1%的琼脂糖凝胶电泳结果显示在470bp和4700bp处各有一条明亮的条带。测序的结果与M1 RNA的DNA模板序列完全一致。 结论:成功构建了特异性抗乙型肝炎病毒C区基因的M1GS RNA核酶的真核表达载体pEGFP-GS,为深入研究M1GS RNA核酶的抗HBV作用奠定了实验基础。
[Abstract]:Aim: to construct the eukaryotic expression vector of M1GS RNA ribozyme specific to the (HBV) C region of hepatitis B virus (HBV) for gene therapy. Methods: PCR primers for DNA template of M1GS RNA ribozyme were designed by using 2333nt of C region of HBV ayw subtype as cleavage site. Using plasmid pTK117 containing DNA sequence encoding M1 RNA as template, the DNA template of M1GS RNA ribozyme specific to HBV C region was obtained by PCR amplification. The recombinant plasmid pEGFP-GS. was obtained from the eukaryotic expression vector pEGFP-C1, which was digested by restriction endonuclease EcoR 鈪，
本文编号：2394063
[Abstract]:Aim: to construct the eukaryotic expression vector of M1GS RNA ribozyme specific to the (HBV) C region of hepatitis B virus (HBV) for gene therapy. Methods: PCR primers for DNA template of M1GS RNA ribozyme were designed by using 2333nt of C region of HBV ayw subtype as cleavage site. Using plasmid pTK117 containing DNA sequence encoding M1 RNA as template, the DNA template of M1GS RNA ribozyme specific to HBV C region was obtained by PCR amplification. The recombinant plasmid pEGFP-GS. was obtained from the eukaryotic expression vector pEGFP-C1, which was digested by restriction endonuclease EcoR 鈪，
本文编号：2394063
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