香蕉乙烯受體基因表達質體之構築及單株抗體之製備

Construction of Expression Plasmids and Preparation of Monoclonal Antibodies for Banana Ethylene Receptor Mh-ERS1

香蕉乙烯受體cDNA pBER1以PstI與HindIII酶切可得到1,191 bp之基因片段，經與載體pQE30進行接合，構築完成的質體經轉型至M15（pREP4）大腸桿菌，以1 mM IPTG（Isopropylthio-β-D-galactoside）誘導生產融合蛋白，可獲得分子量45 kDa之His-tag融合蛋白（fusion protein），以親和層析管柱（affinity chromatography）進行純化，進一步將純化所得之融合蛋白進行製備性電泳分離（preparative gel electrophoresis），可獲得純質之乙烯受體融合蛋白。以此純質蛋白誘導BALB／c小白鼠產生免疫反應，取出其脾臟細胞與NS-1品系之骨髓癌細胞進行細胞融合，再藉由酵素免疫分析法（enzyme immunoassay，EIA）與西方雜交分析進行細胞單株化之篩選，結果獲得七株對乙烯受體具專一性之單株抗體細胞，接著以此單株化細胞注射入小白鼠腹腔中，以誘導BALB／c小白鼠生產含乙烯受體專一性抗體之腹水，再藉由硫酸銨分劃與離心濃縮、透析處理後，得到較高品質之抗體，最後藉由西方轉漬分析，證實已獲得乙烯受體之專一性單株抗體。 Phytohormone ethylene in banana determines the time need for ripening and shelf life. To understand the perception of ethylene in banana, we isolated an ethylene receptor cDNA pBER1 from banana cDNA library. Then we expressed the pBER1 truncated protein in E. coli by pQE30 vector system with the fusion protein exists as insoluble inclusion body. The 45 kDa pQE-P/H polypeptide purified by Ni2+ affinity column and preparative gel electrophoresis was used as an antigen to immunize BALB/c mouse to obtain monoclonal antibody for immunological assay.