Cheng An-Chun, Yu Xiao-Na, Wang Ming-Shu, Zhu De-Kang, Li Ling, Sun Lei, Chen Xiao-Yue
Avian Disease Research Center, College of Animal Science and Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China.
Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):440-5.
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 BamH I/Hind III 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 10(9) PFU 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.
根据GenBank中分离自人体的大肠杆菌的pilA基因序列，使用Oligo 6.0设计一对引物。以从鸭中分离的致病性大肠杆菌为模板，通过PCR获得pilA基因，并将其克隆到pMD18-T载体中。通过PCR、限制性内切酶分析、DNA测序进行鉴定，然后亚克隆到原核表达载体pET-32a(+)的BamH I/Hind III位点，成功构建重组表达质粒pET-32a-pilA。将该质粒转化到大肠杆菌BL21(DE3)中，用IPTG诱导表达36kD的pilA重组蛋白。该蛋白经镍琼脂糖亲和层析纯化，并用弗氏佐剂制成疫苗。分别在1日龄和8日龄时用该疫苗对雏鸭进行免疫。末次免疫两周后，通过ELISA检测鸭血清抗体效价，并用10(9) PFU致病性大肠杆菌GH1.2强毒株对雏鸭进行攻毒。根据死亡率、大肠杆菌再分离率和病理变化等级评估pilA重组蛋白疫苗的免疫保护效果。结果显示，抗体效价为1:12800，而用同源全细胞大肠杆菌灭活疫苗免疫的雏鸭检测到的抗体效价为1:200。免疫雏鸭的死亡率、大肠杆菌再分离率、病理变化程度均低于对照雏鸭，差异显著或极显著(P < 0.01或P < 0.05)，但与用同源全细胞大肠杆菌灭活疫苗免疫的雏鸭相比无显著差异(P > 0.05)。结果表明，pilA重组蛋白对大肠杆菌GH1.2强毒株的攻毒具有一定的免疫保护作用。
