以超氧歧化酶基因作為分子篩選標誌之探討

Transformant selection using superoxide dismutase (SOD) gene

沈佩璇(Pei-Hsuan Shen)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02975

摘要

建構表現載體時，抗藥性基因是最常被應用的篩選標記。近年來異源表達系統蓬勃發展，除了一般基因重組的微生物，其他如基因改造植物(Genetically modified plants, GMP)，也常以抗生素抗性基因當作標示基因，其所攜帶的轉殖基因可能經由花粉傳播，或在土壤造成微生物間基因水平移轉(Horizontal gene transfer)的問題。同時，由於人類過分依賴抗生素，衍生成抗生素濫用情形，造成抗藥性菌株增加，未來許多疾病可能會造成無藥可醫的窘境。因此許多先進國家已嚴格限制，甚至全面禁止抗生素的使用。所以如果能尋找新的替代標示基因來取代抗生素基因，將可解決此問題的發生。
本研究從耐輻射球菌中選殖超氧歧化酶(sod)基因，此基因廣泛存在於好氧生物體中，可催化活性氧物質超氧陰離子，將其歧化成過氧化氫和氧氣。將此sod基因片段建構在分子選殖中常使用的大腸桿菌表達載體pET系統中。再將此載體轉形至E. coli中，利用一種可生成超氧陰離子的化合物 ─ methyl viologen，配合一帶正電性物質CX (Compound X)來篩選出具有sod基因載體的轉形株。進行固態培養基、液態培養基，及同時固態和液態條件的篩選，並模擬轉形條件及實際轉形實驗測試，結果顯示在特定菌數下，可經由液態和固態培養基篩選出帶有sod質體菌株。利用此篩選方式顯示sod基因可作為一篩選標記替代抗藥性基因，可避免使用抗生素。

關鍵字

超氧歧化酶超氧陰離子 ； methyl viologen ； 耐輻射球菌

並列摘要

To construct expression vector, antibiotic resistance gene is the most used selectable marker. Recently, the heterologous expression system has been developed extraordinarily. The antibiotic resistance gene is often useful in enabling not only genetic recombination microorganism but also genetically modified plants (GMP). However, there are some problems about widespread occurrence of transgenes in ecosystems dispersed through pollen or horizontal gene transfer in the soil microorganism. Meanwhile, because human rely on antibiotics excessively , it is in increasing antibiotics resistance strains that the misuse of antibiotics phenomenon results. The serious effect will lead no drugs to treat disease in the future. For this reason, many developed countries have limited or even prohibited the use of antibiotics. It is necessary to look for a new selectable gene to replace for antibiotic resistance gene.
The goal of this study is to establish a plasmid with sod gene which is cloned from Deinococcus radiodurans. All of aerobic organism have the sod gene whose protein product can catalyze superoxide anion to hydrogen peroxide and oxygen. First, the sod gene was inserted into a E. coli expression vector- pET and then the pET-SOD vector was transformed into E. coli. Methyl viologen which produced superoxide anion and a positive chemical CX was used to screen whether transformant had sod gene vectors. The method of screen included solid medium only, broth medium only and both solid and broth medium. The result showed that E. coli with sod plasmid could be selected in specific cell numbers through both solid and broth medium screen. This method can be a new approach to replacement for antibiotic resistance gene.

並列關鍵字

superoxide dismutase ； selectable marker,

參考文獻

60. 王澤嫻。2008。以微脂體或微胞包埋血紫質之光動力殺菌探討。國立台

63. 羅致述，陳淑娟。2005。行政院農業委員會農業藥物毒物試驗所技術專刊第136號。基改植物抗藥性基因移轉與安全評估。

1. Abreu, I. A., A. Hearn, H. An, H. S. Nick, D. N. Silverman, and D. E. Cabelli. 2008. The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations. Biochemistry 47:2350-6.

3. Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411-21.

4. Battista, J. R. 1997. Against all odds: the survival strategies of Deinococcus radiodurans. Annu Rev Microbiol 51:203-24.