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以 pPICZαB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 6 9kD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D2VE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D2VE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。
The pPICZαB vector was used to clone the full-length E gene from C6 / 36 diseased cells infected with D2V by RT-PCR. The recombinant plasmid was integrated into Pichia pastoris by electroporation. After antibiotic screening, phenotyping and PCR The multi-copy integrative bacteria of Mut + type were obtained and the fusion protein of 6 9 kD was induced by methanol induction, which was consistent with the theoretical value of the molecular weight of D2V envelope glycoprotein containing histidine tail. Immunoblotting confirmed that the expression product could be associated with D2VE specificity Monoclonal antibody and D2V polyclonal antibody were reacted. The expressed product was purified by metal chelate affinity chromatography to obtain the purified histidine-tailed E fusion protein and retain its immunoreactivity. Studies have shown that the cloned full-length D2VE gene can be highly secreted expression in Pichia pastoris, E fusion protein maximum expression of 0 1g / L.