利用2A胜肽於嗜甲醇酵母菌Pichia pastoris建立無藥篩選系統

Abstract

基因改造微生物和轉基因植物常以抗生素抗性基因或殺草素抗性基因當作篩選標記，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。本研究欲結合2A胜肽、綠色螢光蛋白質基因及流式細胞儀於嗜甲醇酵母菌Pichia pastoris建立無藥篩選系統。2A胜肽係介於口足疾病病毒(food and mouth disease virus, FMDV) 結構蛋白質與複製蛋白質之間，一段由16至20個胺基酸組成的胜肽。FMDV基因體由單一啟動子同時轉錄出多基因融合的mRNA，再共轉譯出多蛋白融合產物，在後轉譯修飾時期辨識2A胜肽C-端Glycine與下游2B胜肽N-端proline的位置，進行自我斷裂(self-cleavage)形成多個蛋白質產物，此過程稱為2A-mediated cleavage，已證實可在大多數真核生物共表現多個蛋白質。本研究以AOX1為啟動子，並以2A胜肽將GUS和綠色螢光蛋白質(EGFP)基因融合(fusion)成一雙順反子(bicistronic)表現質體。先利用抗生素篩選轉形株，胞外上清液進行蛋白質電泳分析及GUS活性測定;轉形酵母菌細胞以螢光顯微鏡和螢光激活細胞分選(fluorescent-activated cell sorting) 分析綠色螢光表現量。刪除抗藥性基因的質體在轉形至P. pastoris後以螢光激活細胞分選儀(fluorescent-activated cell sorter)篩選出帶有EGFP表現的轉形株。這些轉形株再重新以甲醇誘導蛋白質表現，結果顯示GUS活性與EGFP表現量成正比，驗證了2A胜肽前後蛋白質在同一雙順反子表現載體中具有同比例的蛋白質表現量。此無藥篩選系統利用螢光激活細胞分選儀預測綠色螢光表現量高的轉形株，提供後續蛋白質誘導表現一可信的參考，唯分選前甲醇誘導時間還可縮短以達高通量篩選的目的。

Antibiotic and herbicide resistance genes have been commonly used as selectable markers in genetically modified microorganisms and transgenic plants. However, problems with the widespread use of transgenes arise when they are dispersed throughout an ecosystem through either pollen or horizontal gene transfer by soil microorganisms. To partially alleviate this problem, we sought to develop a drug-free selection system in the methylotrophic yeast Pichia pastoris. To do so, we constructed a vector for transgenic protein expression that harnesses both the reporter gene egfp and 2A-mediated cleavage. As a proof of principle, a bicistronic expression vector was constructed with gus and egfp linked by a 2A peptide cloned downstream of the AOX1 promoter. In our first construct, we included an antibiotic resistance gene and screened transformants by antibiotics. Extracellular supernatants were analyzed by protein electrophoresis and GUS assay. EGFP expression was analyzed by fluorescent microscopy and fluorescent-activated cell sorting (FACS). Once it was shown that our 2A-mediated cleavage was successful and that both GUS and EGFP were properly expressed, we constructed the same vector, but without the antibiotic resistance gene. After transformation of the antibiotic marker-free plasmid into P. pastoris, we screened for transformants by EGFP expression with FACS. The transformants selected by FACS cell sorter demonstrate that GUS activity and EGFP expression level has positive correlation, which proves the hypothesis of equimolar expression of protein upstream and downstream of 2A peptide in bicistronic vector.