【摘要】：RNA干擾(RNA interference,RNAi)是指雙鏈RNA特異性地誘發(fā)與其序列同源的mRNA分子被降解,從而抑制基因表達(dá)的現(xiàn)象,是一種特殊的轉(zhuǎn)錄后基因表達(dá)沉默(post transcriptional gene silence,PTGS)現(xiàn)象。其高效專一地抑制特定基因表達(dá)沉默的特點(diǎn)使RNA干擾技術(shù)被廣泛應(yīng)用于基因功能研究。近年來在細(xì)胞和動(dòng)物體系中進(jìn)行的抗腫瘤和抗病毒等應(yīng)用研究表明,RNA干擾技術(shù)在疾病治療領(lǐng)域同樣具有誘人的應(yīng)用前景。然而要將該技術(shù)成功應(yīng)用于臨床實(shí)踐,仍有幾個(gè)關(guān)鍵問題需要解決,其中之一是如何以較低的成本大規(guī)模制備siRNA。本論文的工作針對(duì)這一關(guān)鍵問題提出了一種新的siRNA制備方法。并成功利用以該方法制備的siRNA抑制了乙型肝炎病毒(HBV)在人肝癌細(xì)胞以及小鼠肝臟內(nèi)的復(fù)制。 第一章中的工作建立了利用大腸桿菌表達(dá)并純化目的基因雙鏈RNA的方法。其方法是構(gòu)建了帶有雙向T7和tac啟動(dòng)子的表達(dá)載體,然后將目的基因片段插入到位于兩個(gè)啟動(dòng)子之間的多克隆位點(diǎn)中。得到的重組質(zhì)粒能夠在大腸桿菌宿主菌中高效表達(dá)雙鏈RNA。通過改變誘導(dǎo)時(shí)的菌液濃度以及誘導(dǎo)時(shí)間優(yōu)化了發(fā)酵條件。發(fā)酵菌體中的雙鏈RNA通過CF-11親和層析柱純化得到。 第二章中的工作包括制備大腸桿菌Ⅲ型RNA水解酶并優(yōu)化其水解雙鏈RNA的條件以及建立用離子交換層析和分子篩層析分離純化水解得到小片段RNA的方法。N端帶六個(gè)組氨酸標(biāo)記的大腸桿菌Ⅲ型RNA水解酶(His-RNase Ⅲ)利用pET-30a表達(dá)載體得到表達(dá),通過Ni~(2+)螯合親和層析柱進(jìn)行純化。在將水解反應(yīng)體系中的Mg~(2+)用Mn~(2+)替代以后,His-RNase Ⅲ可以將雙鏈RNA水解為以21 bp為主的小片段RNA,稱為esiRNA。反應(yīng)體系中His-RNase Ⅲ和底物雙鏈RNA的用量以及Mn~(2+)濃度三者之間必需保持合適的比例,改變?nèi)魏我环N因素都會(huì)影響esiRNA的得率。水解混合物中的esiRNA通過DEAE樹脂離子交換層析以及分子篩層析得到純化。在第四章中實(shí)現(xiàn)了裂殖酵母Dicer羧基端RNase Ⅲ結(jié)構(gòu)域在大腸桿菌中的表達(dá),實(shí)驗(yàn)數(shù)據(jù)表明它能夠在體外反應(yīng)中將長雙鏈RNA水解成21bp左右的siRNA,但是其表達(dá)量不高,酶活性偏低,不適合用于siRNA的大規(guī)模制備。 第三章中的工作首先驗(yàn)證了用新方法制備的esiRNA能夠在哺乳動(dòng)物細(xì)胞中特異而高效地抑制同源基因的表達(dá)。在SMMC7721和293T細(xì)胞中,綠色熒光蛋白的表達(dá)被同源的esiRNA特異性地抑制。在此基礎(chǔ)上,針對(duì)HBV基因重疊的特點(diǎn)和病毒DNA序列的保守性,用軟件分析預(yù)測(cè)選定位于S基因和X基因編碼序列之間的HBV聚合酶編碼序列來制備esiRNA(siHBVP)。在HepG2細(xì)胞中,siHBVP劑
[Abstract]:RNA interference (RNA interference,RNAi) is a special post-transcriptional gene expression silencing phenomenon in which double-stranded RNA specifically induces the degradation of homologous mRNA molecules and inhibits gene expression. RNA interference technique has been widely used in gene function research because of its high efficiency and specificity in suppressing specific gene expression silencing. In recent years, antitumor and antiviral studies in cell and animal systems have shown that RNA interference technology has the same attractive application prospects in the field of disease treatment. However, there are still several key problems to be solved for the successful application of this technique in clinical practice. One of the key problems is how to prepare siRNA. on a large scale at low cost. In this paper, a new preparation method of siRNA is proposed to solve this key problem. The siRNA prepared by this method successfully inhibited the replication of hepatitis B virus (HBV) in human hepatoma cells and mouse livers. In the first chapter, a method for expression and purification of double stranded RNA of target gene by E. coli was established. The expression vector with bidirectional T7 and tac promoters was constructed and the target gene fragment was inserted into the polyclonal site between the two promoters. The recombinant plasmid can efficiently express double-stranded RNA. in Escherichia coli. The fermentation conditions were optimized by changing the concentration of bacteria solution and the induction time. The double stranded RNA in fermentation bacteria was purified by CF-11 affinity chromatography. The work in chapter 2 includes the preparation of Escherichia coli type 鈪，

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