鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

Prokaryotic Expression of pilA Gene of Type Ⅰ Pilus of Enteropathogenic Escherichia coli Isolated from Ducks and the Immunoprotection Effect of Recombinant Expression Protein Challenged with Virulent Strains

摘要:

根据GenBank中人源大肠杆菌pilA基因序列，用OLIGO6.0设计PCR引物，从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体，经PCR、酶切和DNA测序鉴定后，将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a（+）的BamHⅠ和HindⅢ位点间，成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21（DE3），用IPTG诱导，表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化，与等量弗氏佐剂混合制备pilA重组蛋白疫苗，分别在1日龄、8日龄时两次对雏鸭进行免疫，二免后2周测定鸭血清中的ELISA抗体效价，并以10⁹PFU同源菌株GH1.2攻毒，根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800，全菌灭活苗免疫组的血清ELISA抗体效价为1∶200；同源菌株攻毒后，pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著，与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果

Abstract:

A pair primer was designed by Oligo 6.0 according to the pilA gene sequence of E. coli isolated from human in GenBank. The pilA Gene was obtained by PCR with the enteropathogenic E.coli isolated from ducks as template and cloned into pMD18-T vector. It was identified by PCR, restriction endonuclease analysis, DNA sequencing and then subcloned into BamHⅠ/HindⅢ site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-pilA was constructed successfully. The plasmid was transformed into Eschericha coli BL21 (DE3) and 36kD pilA recombinant protein was expressed be induced with IPTG. The protein was purified by Ni-agarose affinity chromatograghy and was prepared as vaccine with Freund's adjuvant. The ducklings were immunized with the vaccine at 1 and 8-day-old respectively. Two weeks after last immunized, the antibody titer of duck serum was detected by ELISA and the ducklings were challenged with 10⁹PFU enteropathogenic E. coli GH1.2 virulent strain .The immunoprotection effect of pilA recombinant protein vaccine was evaluated according to the mortality, re-isolated rate of E. coli, and grades of pathological changes. The results show that the antibody titer are 1∶12800, but 1∶200 were detected from ducklings immunized with homologous whole cells E. coli inactivated vaccine.The mortality, re-isolated rate of E. coli, degree of pathological changes of immunized ducklings is lower than that of the control ducklings and showed significant or extremely significant differences（P＜0.01 or P＜0.05）, but non-significant difference compared to the ducklings which immunized with homologous whole cells E. coli inactivated vaccine（P＞0.05）. The results show that pilA recombinant protein has some immunoprotection effect with the challenging of virulent strains of E. coli GH1.2.