【摘要】：目的:構(gòu)建特異性抗乙型肝炎病毒(HBV)C區(qū)基因的M1GS RNA核酶的真核表達(dá)載體,用于乙型肝炎的基因治療研究。 方法:選擇HBV ayw亞型C區(qū)基因2333nt為切割位點,設(shè)計M1GS RNA核酶的DNA模板的PCR引物。以含有編碼M1 RNA的DNA序列的質(zhì)粒pTK117為模板,通過PCR擴增得到特異性抗HBV C區(qū)基因的M1GS RNA核酶的DNA模板,并將其和真核表達(dá)載體pEGFP-C1經(jīng)限制性核酸內(nèi)切酶EcoR Ⅰ和Sal Ⅰ分別雙酶切后回收,再行連接反應(yīng),得到重組質(zhì)粒pEGFP-GS。轉(zhuǎn)化大腸桿菌JM109,選取陽性克隆堿裂解法進(jìn)行質(zhì)粒抽提,Sal Ⅰ和EcoR Ⅰ雙酶切電泳鑒定和測序鑒定。 結(jié)果:重組質(zhì)粒pEGFP-GS經(jīng)EcoR Ⅰ和Sal Ⅰ酶切后,酶切產(chǎn)物行1%的瓊脂糖凝膠電泳結(jié)果顯示在470bp和4700bp處各有一條明亮的條帶。測序的結(jié)果與M1 RNA的DNA模板序列完全一致。 結(jié)論:成功構(gòu)建了特異性抗乙型肝炎病毒C區(qū)基因的M1GS RNA核酶的真核表達(dá)載體pEGFP-GS,為深入研究M1GS RNA核酶的抗HBV作用奠定了實驗基礎(chǔ)。
[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，

本文編號：2394063