用β-半乳糖苷酶报告基因定量测定E.coli体内硒代半胱氨酸的掺入效率

发布时间：2018-08-25 16:31

【摘要】：硒是人体必需的微量元素之一。在生物体内,它主要是通过构成含硒酶来发挥作用。随着对谷胱甘肽过氧化物酶研究的深入,研究人员发现硒是GPX催化反应的必要组分,且以硒代半胱氨酸(selenocysteine, Sec)的形式作为酶的催化基团发挥作用。令人感兴趣是,Sec是由终止密码子UGA编码,以共翻译的形式插入到新生肽链中,又被称为第21种氨基酸。因此,Sec的掺入机制不同于正常氨基酸,在原核生物中涉及一个顺式作用元件和四个基因产物。在Escherichia coli中,Sec的表达效率只有正常氨基酸的1-3%,所以硒蛋白的体内表达量非常低。在本文工作中,我们研究selA,selB和selC基因对硒代半胱氨酸通读效率的影响,进而探讨硒蛋白在大肠杆菌中的表达效率。 因为selA,selB和selC基因都参与了硒代半胱氨酸的掺入过程,因此我们考察了三者单独或者一起与硒蛋白基因共表达时,Sec通读效率的变化。实验结果表明,与Sec单独表达时相比,当与selC基因共表达时,Sec的通读效率增加了40%。而当与selA或selB基因共表达时,Sec的通读效率分别下降了39%和89%。这表明selA和selB基因不但没有促进反而抑制了Sec的通读。而当与selB和selC基因共表达时,Sec的通读效率增加了11%,在此基础上增加selA基因的表达,通读效率降低了29%。降低阿拉伯糖诱导剂的量后,selAB和selC基因共表达使Sec的通读效率增加了19%。因此,我们推测SelA是一种起负向调节作用的蛋白,SelB是一种起关键性调节作用的蛋白,它可以在一定程度上抑制SelA蛋白的表达水平,同时也由于自身的过量导致翻译的终止加强,Sec通读效率的大幅度下降。另外,SelB蛋白也可能在蛋白质翻译水平抑制了翻译的起始。当我们改用目前世界上表达硒蛋白普遍通用的条件时,共表达selAB,selC基因的Sec掺入效率提高了5.46倍,而共表达pSUABC质粒的只提高了2.2倍。我们的方法要明显优于采用pSUABC质粒的方法,这更有利于Sec的掺入,也更能促进硒蛋白的表达。
[Abstract]:Selenium is one of the essential trace elements in human body. In organisms, it works mainly by making up selenium-containing enzymes. With the further study of glutathione peroxidase, the researchers found that selenium is an essential component in the catalytic reaction of GPX and acts as the catalytic group of the enzyme in the form of selenocysteine (selenocysteine, Sec). Interestingly, SEC is encoded by the termination codon UGA and inserted into the neopeptide chain in the form of cotranslation, also known as the 21st amino acid. Therefore, the incorporation mechanism of Sec is different from that of normal amino acids and involves a cis-acting element and four gene products in prokaryotes. The expression efficiency of SECs in Escherichia coli is only 1-3 of that of normal amino acids, so the expression of selenoprotein is very low in vivo. In this work, we studied the effect of selA,selB and selC genes on the reading efficiency of selenocysteine, and then studied the expression efficiency of selenoprotein in Escherichia coli. Since both selA,selB and selC genes are involved in the incorporation of selenocysteine, we investigated the changes in the reading efficiency of selenocysteine when the three genes were co-expressed alone or together with selenoprotein genes. The results showed that compared with Sec alone, the reading efficiency of SECs increased by 40% when co-expressed with selC gene. When co-expressed with selA or selB gene, the reading efficiency of SEC decreased by 39% and 89%, respectively. This indicated that selA and selB genes not only did not promote but inhibited Sec reading. When co-expressed with selB and selC genes, the reading efficiency of SEC increased by 11%, and the expression of selA gene increased on this basis, and the reading efficiency decreased by 29%. After reducing the amount of arabinose inducer, the co-expression of Sec and selC gene increased the reading efficiency of Sec by 19%. Therefore, we speculate that SelA is a protein that plays a negative role in regulating the expression of SelA protein. At the same time, due to their own excess, the termination of translation to strengthen the efficiency of Sec reading. In addition, SelB protein may inhibit the initiation of translation at the level of protein translation. When we switched to the current universal conditions for the expression of selenoprotein in the world, the Sec incorporation efficiency of co-expression of selAB,selC gene increased 5.46 times, while that of co-expressed pSUABC plasmid increased only 2.2 times. Our method is better than that of pSUABC plasmid, which is more favorable to the incorporation of Sec and can promote the expression of selenoprotein.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

【参考文献】

相关期刊论文 前5条

1 谢明,吴淑华,栾向红,徐云鹤,万晓余,潘卫,侯云德;大肠杆菌M增强子样序列结构和功能的研究[J];中国科学C辑:生命科学;1997年02期

2 蒋志华,牟颖,李维佳,阎岗林,罗贵民;生物合成硒蛋白机制的研究进展[J];生物化学与生物物理学报;2002年04期

3 曹诚,石成华,李平,马清钧;应用CTB基因启动子及信号肽序列构建分泌性表达系统[J];生物工程学报;1995年02期

4 曹诚,石成华,李平,马清钧;霍乱毒素B亚基基因具有自己的启动子[J];生物技术通讯;1996年03期

5 王晓丹,杨天忠,徐钤,彭朝辉,侯云德,颜子颖;质粒型单纯疱疹病毒载体的构建及其在真核细胞中的表达[J];中华微生物学和免疫学杂志;1997年04期

，

本文编号：2203474