整合位置对基因在大肠杆菌染色体上表达的影响

发布时间：2018-04-30 18:06

  本文选题：Red同源重组 + 基因表达　； 参考：《安徽大学》2017年硕士论文

【摘要】：大肠杆菌是已知的最为清楚的模式生物菌种之一,它的染色体结构以及基因组序列都已得到完整的解析,并且由于其操作简单,繁殖快等优点,成为了基因工程中常用的宿主菌。基因在染色体上的位置决定了基因的特性,改变基因的位置可能会改变宿主的基本特征或基因的表达情况。本文拟通过将报告基因整合到染色体上不同位置,研究整合位点对基因表达的影响。λ噬菌体的Red重组系统已经成为基因工程实验中改造的重要工具,由于其方便、高效等特性,已广泛用于大肠杆菌染色体基因整合、替换及基因敲除等。大肠杆菌基因组存在非必须区域,删除这些序列对生物体的稳定性以及基因的表达没有影响。因此本课题利用Red同源重组系统,将报告基因lacZ整合到大肠杆菌染色体上并替换掉非必须区域,研究染色体的位置对基因表达的影响。在大肠杆菌DH1染色体OriC两侧选取相对对称的10个非必须区域,作为报告基因lacZ整合位点。通过两步双链断裂促进同源重组技术实现报告基因在不同位点的整合。该方法需要构建两个供体质粒并通过化转方式导入到细胞内,完成两步同源重组过程。第一步:共转供体质粒p15AD2IC-XLR和辅助质粒至DHl-qlacZ中,L-阿拉伯糖诱导辅助质粒表达Red重组酶和Ⅰ-CreⅠ归巢内切酶,Ⅰ-CreⅠ归巢内切酶可切割供体质粒p15AD2IC-XLR释放Cm抗性基因,在Red重组酶的作用下,Cm筛选标记基因替换染色体上非必须区域基因,完成第一步同源重组。第二步:将第二步供体质粒pBRIS-CP6lacZ和辅助质粒共同转化到第一步阳性菌中,通过加入L-阿拉伯糖诱导表达Ⅰ-SecⅠ归巢内切酶和Red重组酶,Ⅰ-SecⅠ归巢内切酶可识别供体质粒上Ⅰ-SecⅠ序列,释放打靶片段CP6lacZ,在Red重组酶的作用下同源重组替换Cm抗性片段,将报告基因lacZ整合到大肠杆菌染色体上指定区域,通过菌液PCR鉴定,最终获得宿主菌DHl-XCP6lacZ。通过两步同源重组共获得10个实验菌株,对这10个实验菌进行表达培养,根据β-半乳糖苷酶可与ONPG反应生成黄色物质,用以检测lacZ基因的表达水平,再根据实时荧光定量PCR原理,检测不同实验菌株中lacZ基因的转录水平和基因拷贝数,所有的实验结果均显示:基因位置越靠近复制起始位点,基因的表达水平、转录水平、拷贝数就越高,并且此结果不受基因方向的影响。为了考察该结论是否具有普遍适用性,我们将Para-T7启动子控制表达的抗VEGF抗体Fab片段基因onHpL整合到染色体上不同位点进行表达研究。ELISA检测Fab片段的表达情况,其结果符合预期,再次验证了前面结论。
[Abstract]:Escherichia coli is one of the most well known model organism species. Its chromosome structure and genome sequence have been completely analyzed, and because of its simple operation, rapid reproduction and other advantages, It has become a common host bacteria in genetic engineering. The location of the gene on the chromosome determines the characteristics of the gene. Changing the location of the gene may change the basic characteristics of the host or the expression of the gene. This paper intends to study the effect of integration sites on gene expression by integrating the reporter gene into different chromosomes. The Red recombination system of 位 phage has become an important tool in genetic engineering experiments, because of its convenience, high efficiency and other characteristics. It has been widely used in Escherichia coli chromosome gene integration, substitution and gene knockout. Escherichia coli genomes have non-essential regions, and deletion of these sequences has no effect on the stability of organisms and gene expression. Therefore, using Red homologous recombination system, the reporter gene lacZ was integrated into Escherichia coli chromosome and replaced by non-essential region, and the effect of chromosome location on gene expression was studied. Ten non-essential regions of relative symmetry were selected on the two sides of the OriC of E. coli DH1 chromosome as the lacZ integration site of the reporter gene. Two-step double strand breaks were used to promote homologous recombination to achieve the integration of the reporter gene at different sites. In this method, two donor plasmids were constructed and introduced into the cells by chemical transformation to complete the two-step homologous recombination process. The first step was to co-transfer donor plasmid p15AD2IC-XLR and auxiliary plasmid into DHl-qlacZ to induce the expression of Red recombinant enzyme and 鈪，

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