腐蹄病节瘤拟杆菌纤毛蛋白与绵羊白细胞介素-2融合基因表达载体的构建及表达

Construction of a Plasmid pmPili IL-2 Expressing a Fusion Construct of D.nodosus Pili Gene and Ovine IL-2 Gene

根据腐蹄病节瘤拟杆菌纤毛蛋白基因 (Pili基因 )序列 ,在 Pili基因 6 19bp Ehe 酶切位点上插入绵羊白细胞介素 - 2基因 (Ovi IL- 2基因 ) ,构建了 Pili基因与 Ovi IL- 2基因的融合基因表达载体。先用 Eco R 、Bam H 双酶切p Bluescript- Ovi IL - 2重组质粒 ,获得两端具有 Eco R 、Bam H 位点的 Ovi IL - 2基因片段 ,用 Klenow补平后 ,与经Ehe 酶切后的重组质粒 PME2 90 - Pili进行平端连接 ;转化 JM10 5感受态细胞 ,筛选阳性克隆 ,分别用 Bam H 、Eco R 、Xba 、Bgl 、Pvu 等进行酶切鉴定。然后将阳性重组质粒转染 BL - 2 1表达宿主感受态细胞 ,用 0 .1m ol/ L Mg Cl2沉淀法和超声波裂解法提取 BL- 2 1表达 Pili基因与 Ovi IL- 2基因融合基因表达蛋白。对流免疫电泳对重组融合蛋白的特异性分析证明 ,融合蛋白主要在宿主菌体中表达 ;SDS- PAGE电泳结果表明 ,重组融合蛋白分子大小约为 330 0 0 ,表达产物的量约占菌体的 5 .49%。

A fusion expression plasmid was constructed by inserting the ovine IL-2 gene into PME290-Pili in the Pili gene at downstream 619 bp.The plasmid harbouring ovine IL-2 sequence was designated pmPili IL-2.The fusion protein was expressed by transforming the pmPili IL-2 into the competent host cell BL-21(DE3).Specific reaction between PiliIL-2 fusion protein and the antiserum of D.nodosus serotype E Pili was observed by cross electrophoresis.The molecular weight of the recombinant fusion protein was about 33 000 by SDS-PAGE and analyzed with thin layer.The recombinant Pili IL-2 fusion gene was expressed at higher level with about 5.49% of the total proteins of the transformant host cell BL-21(DE3).