腐蹄病节瘤拟杆菌纤毛蛋白的基因克隆与表达

Cloning and Expression of Dichelobacter nodosus Fimbrial Subunit Gene in Pseudomonas aeruginosa

用 PCR技术从腐蹄病 E型节瘤拟杆菌克隆出具免疫保护性抗原 0 .93 kb纤毛蛋白基因 ( Pili基因 ) ,利用该基因构建了纤毛蛋白基因表达载体。先将 PCR产物 Pili基因克隆于 TA Cloning System,扩增后 ,用Eco R 酶切 ,低熔点胶回收 ,经 Klenow补平后 ,用 T4DNA连接酶与中间载体 p PLλ连接 ,将 Pili基因克隆于 p PLλ载体 ;经 Bam H 、Bgl 、Pvu 和 Smal +Bgl 酶切鉴定和 p PLλ-Pili重组质粒 Pili基因序列测定正确后 ,扩增 p PLλ-Pili重组质粒 ,用 Bam H 酶切出 2 .1 kb大小的片段 ,回收后 ,与 p ME2 90表达质粒连接 ,转染宿主细胞 PAK/2 pfs,在营养肉汤中进行 Pili基因的表达。培养 1 8～ 2 4 h后 ,离心 ,向上清液中加入 0 .1mol/L Mg Cl2 提取重组纤毛蛋白。用羊抗兔 E型节瘤拟杆菌抗血清与提取的重组纤毛蛋白进行对流免疫电泳 ,证明重组纤毛蛋白具有特异性 ;染料结合法测定 1 0 0 0 m L上清液中表达纤毛蛋白粗含量约为 1 1 4mg。SDS-PAGE测定重组纤毛蛋白的表达量占总菌量的 1 1 .8%,Western

The fimbrial subunit(pili) gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serotype E by PCR. An expression plasmid was constructed by cloning the pili gene into pME290.The plasmid harbouring the pili sequence was designated pME290 Pili. The pME290 Pili was transformed into the host competent cell PAK/2pfs and the recombinant Pili was expressed in the supernatant of the cultures of the transformant cell PAK/2pfs. The recombinant pili was purified by MgCl 2 from the supernatant of the culture of the transformed PAK/2pfs. The specific reaction of the recombinant pili and antiserum of D.nodosus serotype E pili was demonstrated by cross electrophoresis. The recombinant pili was expressed at high level in PAK/2pfs and amounted to 11.8% of the total protein of the transformed host cell PAK/2pfs.