以pPICZ a B为载体，应用RT—PCR从感染D2V的C6／36病变细胞中克隆全长E基因，电转化法将重组质 粒整合入巴斯德毕赤氏酵母菌，经抗生素筛选、表型鉴定和PCR分析得到Mut 型的多拷贝整合菌，经甲醇诱导培 养可产生69kD的融合蛋白，与含组氨酸尾的D2V包膜糖蛋白分子量理论值相符；免疫印迹证实该表达产物可与 D2V E特异性单抗和D2V 多抗进行反应；表达产物经金属螯合亲和层析可获得纯化的含组氨酸尾的E融合蛋白 并保留其免疫反应性。研究显示克隆的全长D2V E基因可在毕赤氏酵母菌中高效分泌表达，E融合蛋白最大表达 量0．1g／L。

Abstract：The E gene of dengue一2 virus was amplified using RT—PCR mothod from the C6／36 cells in— fected by D2V NGC strain and inserted into pPICZ a B vector．The recom binant plasm id was integret— ed into Pichia pastoris X一33 by electroporation and the expressed products were analyzed w ith SDSPAGE and W estern blotting．High level secreted expression was performed by determining the M ut phenotype and screening m ulti—copy integrants in the recombinant Pichia strains．The molecular m ass of recombinant E glycoprotein was approx． 69kD and secreted into supernatants when induced with methano1．The expression product was able tO reacted with D2V polyclone antibody and D2V E specif— ic M cAb．M CAC purified E—hisidine—tagged protein from the expressed product w ith HisTrap Kit can bind im m unologically tO H is antibody and D2V E specific M cAb in W estern—blot as ay． This study suggests the entire E gene coding D2V envelope glycoprotein can be expressed efficiently in Pichia pastoris and the expressed products of E fusion protein could amount to 0．1 g／L．