摘要
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 E gene of dengue 2 virus was amplified using RT PCR mothod from the C6/36 cells infected by D2V NGC strain and inserted into pPICZ α B vector. The recombinant plasmid was integreted into Pichia pastoris X 33 by electroporation and the expressed products were analyzed with SDS PAGE and Western blotting. High level secreted expression was performed by determining the Mut phenotype and screening multi copy integrants in the recombinant Pichia strains. The molecular mass of recombinant E glycoprotein was approx. 69kD and secreted into supernatants when induced with methanol. The expression product was able to reacted with D2V polyclone antibody and D2V E specific McAb. MCAC purified E hisidine tagged protein from the expressed product with HisTrap Kit can bind immunologically to His antibody and D2V E specific McAb in Western blot assay. 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 0.1g/L.
出处
《中国病毒学》
CSCD
2002年第3期198-201,共4页
Virologica Sinica
基金
广东省科技厅自然科学基金资助课题 (2 0 10 3 6)
关键词
登革2型病毒
E蛋白
酶母菌
分泌表达
Dengue virus
E glycoprotein
Secreted expression
Pichia pastoris
