利用基因重組技術表達大腸桿菌腺嘌呤醣基解酶(MutY)及其包涵體之研究

THUIR > College of Engineering > Department of Chemical and Materials Engineering > Theses and Dissertations > Item 310901/31528

Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/31528

Title:	利用基因重組技術表達大腸桿菌腺嘌呤醣基解酶(MutY)及其包涵體之研究
Other Titles:	Optimizimg the overexpression of E.coli DNA glycosylase MutY and the characterization of its inclusion body
Authors:	鄒孟妘 ZOU, MENG-YUN
Contributors:	顧野松 GU, YE-SONG 化學工程與材料工程學系
Keywords:	包涵體;大腸桿菌腺嘌呤醣基解酶;基因重組;綠色螢光蛋白;大腸桿菌;蛋白質表達 Inclusion body;MutY;Gene Recombination;EGFP;E.coli;Protein Expression
Date:	2019
Issue Date:	2019-12-16T02:19:13Z (UTC)
Abstract:	DNA的錯配修復在預防以及避免基因突變上佔有重要的地位，任何基因上的突變都可能會導致癌症的發生。鹼基對中的鳥糞嘌呤(Guanine, G)在原核生物以及真核生物中都是非常容易被氧化並形成8-oxoG，此種改變為會將原本的G:C配置轉換成T:A配置，導致不可逆的基因突變。為了避免8-oxoG所造成的傷害，生物體對其有相對應的預防機制，大腸桿菌會使用MutT及MutM和MutY使用鹼基切除修復(Base-excision repair)預防8-oxoG所造成的基因突變。在此篇研究中，藉由基因工程的方式將MutY基因分別連結於pET21a(+)以及pSEI的質體，再轉型進入大腸桿菌中，並藉由IPTG誘導MutY蛋白的產生。在高表達的pET21a(+)系統中會以包涵體的方式進行表達，透過分析包涵體與雜蛋白間的關係和包涵體在變性過程中所產生的變化，發現包涵體的形態可能是由目標蛋白逐漸包覆，且最外層主要為雜蛋白所形成的球狀物。而在含有SUMO的pSEI質體中，MutY蛋白則會以可溶性的方式呈現，而又因為pSEI質體在表達過程中能夠將MutY蛋白帶有EGFP的螢光成分，使其在純化過程中，更能明確地將其純化出來，以利後續的應用。 DNA mismatch repair plays an important role for preventing gene mutation. Any genetic mutation may lead to cancer. In prokaryotes and eukaryotes, guanine is very easy attacked by ROS to form 8-oxoG. The 8-oxoG lesion will lead to convert G:C to T:A and this is irreversible. In order to avoid the damage by 8-oxoG lesion, the organism has a corresponding preventive mechanism. In E.coli, base-excision repair is the major mechanism to remove oxidized guanine by specific DNA glycosylases MutT、MutM and MutY. In this study, we ligase MutY gene to pET21a(+) vector and pSEI vector by genetic recombination, respectively. The plasmids transformed to BL21(DE3), and expressed MutY by IPTG. In the system pET21a(+), the overexpression of protein will lead to the formation of inclusion body. By analyzing the relationship between inclusion bodies and the variation of inclusion body during denaturation, we conjecture the inclusion body may be gradually coated by the target protein and the outer layer is the other protein. Because pSEI plasmid containing the sumo, the MutY protein is soluble. The fusion protein containing EGFP, this characteristic may lead to better purification. The pure fusion protein can facilitatation for large applications.
Appears in Collections:	[Department of Chemical and Materials Engineering ] Theses and Dissertations

Files in This Item:

File	Description	Size	Format
107THU00063012-001.pdf		4001Kb	Adobe PDF	35	View/Open

Loading...