1型鸭疫里默氏杆菌p25蛋白基因(p25)的克隆表达及免疫学活性

Cloning and expression of p25 protein gene(p25) of Riemerella anatipestifer serotype 1 and its detection of immunogenicity

为探讨1型鸭疫里默氏杆菌(Riemerella anatipestifer,RA)p25蛋白的免疫原性,从RA1型基因组DNA中扩增出p25基因全长ORF,生物信息学分析显示其为含有完整保守结构域PRK00110的一未知功能保守蛋白。p25基因经BamHⅠ和HindⅢ双酶切后,插入原核表达载体pET-32a(+),构建重组表达质粒pET-32a(+)-p25。测序正确后,将重组表达质粒pET-32a(+)-p25转入E.coli Rosetta,并成功进行了诱导表达。SDS-PAGE分析表明,表达的重组蛋白与预期大小一致,表达产物主要以可溶性形式存在;Western blotting检测显示,该蛋白具有良好的免疫原性。

In order to study the immunogenicity of p25 protein of Riemerella anatipestifer serotype 1,the whole ORF of p25 gene was amplified from RA serotype 1 genomic DNA.The bioinformatics indicated that p25 gene was an unknown function conserved domain contianing an complete CDD named PRK00110.After the amplified product was digested by endonuclease BamH Ⅰand Hind Ⅲ,the p25 gene was cloned into prokaryotic expression vector pET-32a（＋） and the recombinant expression vector pET-32a（＋）-p25 was constructed.The recombinant expression vector pET-32a（＋）-p25,with the correct sequence,was introduced into E.coli Rosetta and expressed successfully in E.coli Rosetta.SDS-PAGE analysis showed that the MW of recombinant protein was about 45 000 and the recombinant protein was mainly existed as soluble protein.Western blotting indicated that this recombinant p25 protein had good immunogenicity.