腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与表达载体的构建

Cloning and Construction Expressing Vector of D.nodosus serotype C Pili Gene

用 PCR技术从腐蹄病 C型节瘤拟杆菌克隆出具有免疫保护性抗原 0 .85kb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。提取 C型节瘤拟杆菌染色体DNA;用所设计的专一性引物进行 PCR,扩增出 pili基因 ;将 pili基因克隆于 TE载体 ,TE-pili重组质粒 pili基因序列测定结果正确 ,用 Eco R 酶切 ,低熔点胶回收 pili基因片段 ,经 klenow补平后 ,用 T4DNA连接酶将其与中间载体p PLλ连接 ,将 pili基因克隆于 p PLλ载体 ,经Bam H 、Bam H +Hind 、Dral酶切鉴定 pili基因正向插入 p PLλ载体 ;扩增 p PLλ-pili重组质粒 ,用 Bam H 酶切出 2 .1 kb大小片段 ,回收后 ,与 PME2 90表达质粒连接 ,转化宿主细胞PAK/ 2 pfs中 ,对获得的重组质粒用 Bam H 酶切 ,出现 2 .1 kb大小的带 。

The pili gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serorype C by PCR. An expression plasmid was constructed by cloning the pili gene into PME290. The plasmas 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/2fs. The recombinant pili was purified by Mgcl2 from the supernatant of the culture of the transformed PAK/2pfs. The specific reaction of the recombinant pili and antiserum of D.nodosus serotype C pili was demonstrated by cross electrophoresis. The recombinant pili was expressed at high level in PAK/2pfs.