【摘要】：硒是人體必需的微量元素之一。在生物體內(nèi),它主要是通過構成含硒酶來發(fā)揮作用。隨著對谷胱甘肽過氧化物酶研究的深入,研究人員發(fā)現(xiàn)硒是GPX催化反應的必要組分,且以硒代半胱氨酸(selenocysteine, Sec)的形式作為酶的催化基團發(fā)揮作用。令人感興趣是,Sec是由終止密碼子UGA編碼,以共翻譯的形式插入到新生肽鏈中,又被稱為第21種氨基酸。因此,Sec的摻入機制不同于正常氨基酸,在原核生物中涉及一個順式作用元件和四個基因產(chǎn)物。在Escherichia coli中,Sec的表達效率只有正常氨基酸的1-3%,所以硒蛋白的體內(nèi)表達量非常低。在本文工作中,我們研究selA,selB和selC基因?qū)ξ腚装彼嵬ㄗx效率的影響,進而探討硒蛋白在大腸桿菌中的表達效率。 因為selA,selB和selC基因都參與了硒代半胱氨酸的摻入過程,因此我們考察了三者單獨或者一起與硒蛋白基因共表達時,Sec通讀效率的變化。實驗結果表明,與Sec單獨表達時相比,當與selC基因共表達時,Sec的通讀效率增加了40%。而當與selA或selB基因共表達時,Sec的通讀效率分別下降了39%和89%。這表明selA和selB基因不但沒有促進反而抑制了Sec的通讀。而當與selB和selC基因共表達時,Sec的通讀效率增加了11%,在此基礎上增加selA基因的表達,通讀效率降低了29%。降低阿拉伯糖誘導劑的量后,selAB和selC基因共表達使Sec的通讀效率增加了19%。因此,我們推測SelA是一種起負向調(diào)節(jié)作用的蛋白,SelB是一種起關鍵性調(diào)節(jié)作用的蛋白,它可以在一定程度上抑制SelA蛋白的表達水平,同時也由于自身的過量導致翻譯的終止加強,Sec通讀效率的大幅度下降。另外,SelB蛋白也可能在蛋白質(zhì)翻譯水平抑制了翻譯的起始。當我們改用目前世界上表達硒蛋白普遍通用的條件時,共表達selAB,selC基因的Sec摻入效率提高了5.46倍,而共表達pSUABC質(zhì)粒的只提高了2.2倍。我們的方法要明顯優(yōu)于采用pSUABC質(zhì)粒的方法,這更有利于Sec的摻入,也更能促進硒蛋白的表達。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R341

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本文編號：2203474