[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].
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.
Cheng
An-Chun
AC
Avian Disease Research Center, College of Animal Science and Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China.
Yu
Xiao-Na
XN
Wang
Ming-Shu
MS
Zhu
De-Kang
DK
Li
Ling
L
Sun
Lei
L
Chen
Xiao-Yue
XY
D000818:Animals; D000831:Animals, Newborn; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D004372:Ducks / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007114:Immunization; D016133:Polymerase Chain Reaction; D011201:Poultry Diseases / Q000276:immunology* / Q000401:mortality / Q000517:prevention & control; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism; D015996:Survival Rate; D014774:Virulence
D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D011994:Recombinant Proteins; D036781:Fimbriae Proteins