Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

Abstract

Objectives: Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC.

Materials and methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA.

Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC.

Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.

Keywords: Enterotoxigenic Escherichia coli; IgY; Immunization; Recombinant protein.

Figure 1

(a) Electrophoresis of colony PCR of the FanC gene with M13 and specific primers, (Lane 1: FanC gene, lane 2: DNA size marker, lane 3-8: FanC gene, lane 9: DNA size marker, lane 10: negative control). (b) digested pTZ57R/T-FanC plasmid, lane 1: DNA size marker and lane 2: digested pTZ57R/T-FanC)

Figure 2

(a) Electrophoresis of colony PCR of the FanC gene with specific primers and pET T7 primers (lane 1: 1kb DNA size marker, lane 2 -6: FanC gene and lane 7: negative control). (b) Digestion of pET32a (+)-FanC (lane 1: digested pET32a(+)-FanC and lane 2: 1kb DNA size marker)

Figure 3

(a) The results of SDS-PAGE. (lane1: pellet uninduced by IPTG, lane 2, 3 and 4: pellets in 2, 4 and 6 hr after induction, respectively, lane 5: protein size marker, lane 6, 7 and 8: pET32a(+)/E. coli in 0, 2, and 4 hr after induction.(b) Dot-blot analysis of the recombinant protein. Brown dot (A) (E. coli/pET32a (+)/FanC) shows transformed bacteria induced by IPTG. (B)PBS and (C), E. coli/pET32a (+)/FanC before the induction shown as negative control

Figure 4

(a) 12% SDS-PAGE analysis of the protein purification. (Lane 1): the induced cell lysate; (lane 2): washed fraction; (lanes 3, 4, 5, and 6): fractions of the eluted proteins (E1, E2, E3, and E4), (lane 7): protein size marker. (b) SDS-PAGE analysis of the purified IgY antibody

Figure 5

(a) Specific activity of anti-FanC IgY (at 1:2000 dilution) with the recombinant protein in treatment and control groups. (b) Changes in the anti-FanC antibody activity in the egg yolk after the last immunization (weeks)