利用大肠杆菌发酵制备siRNA及其在抑制乙型肝炎病毒复制中的应用
发布时间：2018-09-07 18:08
【摘要】：RNA干扰(RNA interference,RNAi)是指双链RNA特异性地诱发与其序列同源的mRNA分子被降解,从而抑制基因表达的现象,是一种特殊的转录后基因表达沉默(post transcriptional gene silence,PTGS)现象。其高效专一地抑制特定基因表达沉默的特点使RNA干扰技术被广泛应用于基因功能研究。近年来在细胞和动物体系中进行的抗肿瘤和抗病毒等应用研究表明,RNA干扰技术在疾病治疗领域同样具有诱人的应用前景。然而要将该技术成功应用于临床实践,仍有几个关键问题需要解决,其中之一是如何以较低的成本大规模制备siRNA。本论文的工作针对这一关键问题提出了一种新的siRNA制备方法。并成功利用以该方法制备的siRNA抑制了乙型肝炎病毒(HBV)在人肝癌细胞以及小鼠肝脏内的复制。 第一章中的工作建立了利用大肠杆菌表达并纯化目的基因双链RNA的方法。其方法是构建了带有双向T7和tac启动子的表达载体,然后将目的基因片段插入到位于两个启动子之间的多克隆位点中。得到的重组质粒能够在大肠杆菌宿主菌中高效表达双链RNA。通过改变诱导时的菌液浓度以及诱导时间优化了发酵条件。发酵菌体中的双链RNA通过CF-11亲和层析柱纯化得到。 第二章中的工作包括制备大肠杆菌Ⅲ型RNA水解酶并优化其水解双链RNA的条件以及建立用离子交换层析和分子筛层析分离纯化水解得到小片段RNA的方法。N端带六个组氨酸标记的大肠杆菌Ⅲ型RNA水解酶(His-RNase Ⅲ)利用pET-30a表达载体得到表达,通过Ni~(2+)螯合亲和层析柱进行纯化。在将水解反应体系中的Mg~(2+)用Mn~(2+)替代以后,His-RNase Ⅲ可以将双链RNA水解为以21 bp为主的小片段RNA,称为esiRNA。反应体系中His-RNase Ⅲ和底物双链RNA的用量以及Mn~(2+)浓度三者之间必需保持合适的比例,改变任何一种因素都会影响esiRNA的得率。水解混合物中的esiRNA通过DEAE树脂离子交换层析以及分子筛层析得到纯化。在第四章中实现了裂殖酵母Dicer羧基端RNase Ⅲ结构域在大肠杆菌中的表达,实验数据表明它能够在体外反应中将长双链RNA水解成21bp左右的siRNA,但是其表达量不高,酶活性偏低,不适合用于siRNA的大规模制备。 第三章中的工作首先验证了用新方法制备的esiRNA能够在哺乳动物细胞中特异而高效地抑制同源基因的表达。在SMMC7721和293T细胞中,绿色荧光蛋白的表达被同源的esiRNA特异性地抑制。在此基础上,针对HBV基因重叠的特点和病毒DNA序列的保守性,用软件分析预测选定位于S基因和X基因编码序列之间的HBV聚合酶编码序列来制备esiRNA(siHBVP)。在HepG2细胞中,siHBVP剂
[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，
本文编号：2229023
[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，
本文编号：2229023
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