腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与序列分析

Cloning and Sequence Analysis of D.nodosus Serotype C Pili Gene in Pseudomonas

提取C型节瘤拟杆菌染色体DNA。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 ) ,将PCR产物pili基因克隆于TE载体 ,在含X - gal和IPTG的AmpLB平板上 ,挑选白色单一菌落进行质粒的筛选 ,用EcoR1酶切提取重组质粒 ,琼脂糖凝胶电泳 ,出现一条 0 .85kb的条带 ,从而筛选出TE - pili重组质粒。对 pili基因进行序列测定 ,结果与国外报道的 pili基因序列一致。

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 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 C pili was demonstrated by cross electrophoresis. The recombinant pili was expressed at high level in PAK/2pfs.