以抗藥性標誌進行酵母菌載體之改良

Abstract

本論文主旨在於利用抗葯性標誌，以構築酵母菌的載體(vector)及改良其轉形系統(transformation system）。首先測試33株酵母菌對抗生素G418之葯敏程度(durg senitivity)；並檢測內生型2μm質體的分佈情形。其中有3株對G418之抗性高於500 μg/ml，而16株Saccharomyces cerevisiae葯敏程度則均在100?350μg/ml。內生型2μm質體只存在於S. cerevisiae中之12株，其他21株則無。 為建立抗葯性轉形系統，故利用抗葯性標誌G418，及含Tn601/903 kanamycin磷酸化?基因（Kmr）之2μm質體，進行S. cerevisiae M12B (α trp1 ura3 cir＋，G418s：100μg/ml)與野生型S. cerevisiae F2 (cir＋，G418s：300μg/ml)及F4 (cir＋，G418s：100μg/ml)轉形實驗。其轉形株抗葯性均高達1000 μg/ml以上。 欲改良實驗菌株，使其具有trp1，leu2及對G418低抗葯程度三項特性，作為轉形實驗之篩選標誌。因此將S．cerevisiae AH22 (a leu2 his 4 cir＋ G418s：300μg/ml)和S. cerevisiae M12B交配，得到532株營養需求性子代。經遺傳分析，得知酵母菌之G418莉敏基因為染色體上一個基因座(gene locus)所控制，且該基因和染色體Ⅲ，Ⅳ及Ⅴ非同一遺傳連鎖群(linkage group)。在532株子代中，具trp1，leu2及G418s：100μg/ml之菌株有8株，其中以S. cerevisiae TL154-α-s (α trp1 leu2 cir＋ G418s：100μg/ml)對G418之抗性較為穩定，所以作為轉形實驗之接受菌株(recipient strain)。 為探討抗葯性標誌基因對質體穩定性的各種影響，構築9個新質體，與原有的8個質體同時對接受菌株TL154－α-s進行轉形實驗，得到14種轉形株。經測試其質體穩定度後，進行分析比較，得到下列結果：(1)具有兩個標誌基因（TRP1及Kmr）質體所得的轉形株，若以G418作為前培養時，可使質體穩定度較使用營養需求培養基者高出甚多。(2)質體中若具有兩個順向排列之IR2 (invert repeat 2)，可發生定位重組(site specific recombination)，造成質體結構之改變，因而導致標誌基因表現型之分離。當質體僅具一個IR2時，則無此現象。(3)2μm質體之STB區若嵌入Kmr基因，質體的穩定度會降低。 以四種含B型肝炎表面抗原基因的質體pMKS-1，pMKS-1-S，pAS32，pAS32-S之轉形株，測試表面抗原之基因表現，其中pMKS-1之轉形株具有較高之表現頻率。進一步篩選pMKS-1轉形株時，得到七株具有較高表現能力之表面抗原生產酵母菌。

Using a drug resistance determinant to construct 2μm-containing vectors to improve yeast transformation was studied. 33 yeast strains were tested for their sensitivity to antibiotic G418. Most of the strains were not able to grow in G418-containing medium. Meanwhile, as the existance of endogenous 2μm DNA was tested, one third of these strains were found to possess 2μm sequence. Vectors containing Kmr determinant sequence and 2μ-ori were found to be useful for the transformation of those G418-sensitive and 2μm-containing strains. In order to combine transforming markers, TRP1, LEU2 and G418 resistance, in same recipient strain, Saccharomyces cerevisiae AH22 (a leu2, his4, cir+, G418S:300 μg/ml) was crossed with S. cerevisiae M12B (α trpl, ura3, cir+, G418S:100 μg/ml). Eight out of 532 auxotrophic progenies had the characters trpl, leu2 and G418 sensitivity (100 μg/ml). One of which, designated as TL154-α-s (α trpl, leu2, cir+, G418S: 100 μg/ml), was quite stable in the G418 sensitivity test, and served as a recipient strain in the yeast transformation experiments. The relationship among auxotrophy, mating type and G418 sensitivity of 532 progenies was analysed. It was suggested that G418 sensitivity of s. cerevisiae was controlled by one chromosomal gene locus and was not located in the same linkage group as chromosome III, IV and V. To investigate the factors which influence plasmid stability, S. cerevisiae TL154-α-s was transformed with 17 different plasmids including 9 of newly constructed. Three of the following results were obtained: (1) plasmid stability of the transformats having two transforming markers, Trp+ and G418r, when precultured in YPD+G418 was more stable than precultured in yeast minimal medium; (2) intramolecular recombination between two IR2 sequences in a plasmid caused changes of plasmid structure and segregation of marker genes; (3) the STB locus of 2μm DNA inserted by Kmr gene resulted in the decrease of marker gene stability. Using the established yeast transformation system, the production of hepatitis B surface antigen (HBV-sAg) in yeast was examined. Gene expression of HBV-sAg gene in different yeast transformants was shown to be detectable, but in a very unexpected fashion.