發(fā)布時(shí)間：2018-04-30 18:06

  本文選題：Red同源重組 + 基因表達(dá)　； 參考：《安徽大學(xué)》2017年碩士論文

【摘要】：大腸桿菌是已知的最為清楚的模式生物菌種之一,它的染色體結(jié)構(gòu)以及基因組序列都已得到完整的解析,并且由于其操作簡(jiǎn)單,繁殖快等優(yōu)點(diǎn),成為了基因工程中常用的宿主菌�；�因在染色體上的位置決定了基因的特性,改變基因的位置可能會(huì)改變宿主的基本特征或基因的表達(dá)情況。本文擬通過(guò)將報(bào)告基因整合到染色體上不同位置,研究整合位點(diǎn)對(duì)基因表達(dá)的影響。λ噬菌體的Red重組系統(tǒng)已經(jīng)成為基因工程實(shí)驗(yàn)中改造的重要工具,由于其方便、高效等特性,已廣泛用于大腸桿菌染色體基因整合、替換及基因敲除等。大腸桿菌基因組存在非必須區(qū)域,刪除這些序列對(duì)生物體的穩(wěn)定性以及基因的表達(dá)沒(méi)有影響。因此本課題利用Red同源重組系統(tǒng),將報(bào)告基因lacZ整合到大腸桿菌染色體上并替換掉非必須區(qū)域,研究染色體的位置對(duì)基因表達(dá)的影響。在大腸桿菌DH1染色體OriC兩側(cè)選取相對(duì)對(duì)稱的10個(gè)非必須區(qū)域,作為報(bào)告基因lacZ整合位點(diǎn)。通過(guò)兩步雙鏈斷裂促進(jìn)同源重組技術(shù)實(shí)現(xiàn)報(bào)告基因在不同位點(diǎn)的整合。該方法需要構(gòu)建兩個(gè)供體質(zhì)粒并通過(guò)化轉(zhuǎn)方式導(dǎo)入到細(xì)胞內(nèi),完成兩步同源重組過(guò)程。第一步:共轉(zhuǎn)供體質(zhì)粒p15AD2IC-XLR和輔助質(zhì)粒至DHl-qlacZ中,L-阿拉伯糖誘導(dǎo)輔助質(zhì)粒表達(dá)Red重組酶和Ⅰ-CreⅠ歸巢內(nèi)切酶,Ⅰ-CreⅠ歸巢內(nèi)切酶可切割供體質(zhì)粒p15AD2IC-XLR釋放Cm抗性基因,在Red重組酶的作用下,Cm篩選標(biāo)記基因替換染色體上非必須區(qū)域基因,完成第一步同源重組。第二步:將第二步供體質(zhì)粒pBRIS-CP6lacZ和輔助質(zhì)粒共同轉(zhuǎn)化到第一步陽(yáng)性菌中,通過(guò)加入L-阿拉伯糖誘導(dǎo)表達(dá)Ⅰ-SecⅠ歸巢內(nèi)切酶和Red重組酶,Ⅰ-SecⅠ歸巢內(nèi)切酶可識(shí)別供體質(zhì)粒上Ⅰ-SecⅠ序列,釋放打靶片段CP6lacZ,在Red重組酶的作用下同源重組替換Cm抗性片段,將報(bào)告基因lacZ整合到大腸桿菌染色體上指定區(qū)域,通過(guò)菌液PCR鑒定,最終獲得宿主菌DHl-XCP6lacZ。通過(guò)兩步同源重組共獲得10個(gè)實(shí)驗(yàn)菌株,對(duì)這10個(gè)實(shí)驗(yàn)菌進(jìn)行表達(dá)培養(yǎng),根據(jù)β-半乳糖苷酶可與ONPG反應(yīng)生成黃色物質(zhì),用以檢測(cè)lacZ基因的表達(dá)水平,再根據(jù)實(shí)時(shí)熒光定量PCR原理,檢測(cè)不同實(shí)驗(yàn)菌株中l(wèi)acZ基因的轉(zhuǎn)錄水平和基因拷貝數(shù),所有的實(shí)驗(yàn)結(jié)果均顯示:基因位置越靠近復(fù)制起始位點(diǎn),基因的表達(dá)水平、轉(zhuǎn)錄水平、拷貝數(shù)就越高,并且此結(jié)果不受基因方向的影響。為了考察該結(jié)論是否具有普遍適用性,我們將Para-T7啟動(dòng)子控制表達(dá)的抗VEGF抗體Fab片段基因onHpL整合到染色體上不同位點(diǎn)進(jìn)行表達(dá)研究。ELISA檢測(cè)Fab片段的表達(dá)情況,其結(jié)果符合預(yù)期,再次驗(yàn)證了前面結(jié)論。
[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，

本文編號(hào)：1825613