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

Document Type: Original Article


1 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad , Iran

2 Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran

3 Department of Animal Science and Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

4 Department of Veterinary Medicine, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran


Objective(s): 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 ETECinfection in animals and humans.


1. Jay CM, Bhaskaran S, Rathore KS, Waghela SD. Enterotoxigenic K99 Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein. Vet Microbiol 2004; 101:153–160.

2. Altmann K, Pyliotis NA, Mukkur TK. A new method for the extraction and purification of K99 pili from Enterotoxigenic Escherichia coli and their characterization. Biochem J 1982; 201: 505–513.

3. Clarke SC. Diarrhoeagenic Escherichia coli an emerging problem? Diagn Microbiol Infect Dis 2001; 41:93–98.

4. Younis EE, Ahmed AM, El-Khodery SA, Osman SA, El-Naker YF. Molecular screening and risk factors of Escherichia coli and salmonella spp. In diarrheic neonatal calves in Egypt. Res Vet Sci 2009; 87:373-379.

5. Lee JH, Isaacson RE. Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99. Infect Immun 1995; 63:4143-4149.

6. Roosendaal B, Damoiseaux J, Jordi W, de Graaf FK. Transcriptional organization of the DNA region controlling expression of the K99 gene cluster. Mol Gen Genet 1989; 215: 250-256.

7. Kobayashi C, Yokoyama H, Neguyen SA, Kodama Y, Kimata T, Izeki M. Effect of egg yolk antibody on experimental cryptosporidium parvum infection in Scid mice. Vaccine 2004; 23:232-235.

8. Mine Y, Kovacs-Nolan J. Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review. J Med Food 2002; 5:159-169.

9. Vega C, Bok M, Chacana P, Saif L, Fernandez F, Parreño V. Egg Yolk IgY: Protection against Rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves. Vet Immunol Immunopathol 2011; 142:156-169.

10. Bradford MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254.

11. Polson A, von Wechmar, MB, van Regenmortel MH. Isolation of viral IgY antibodies from yolks of immunized hens. Immunol Commun 1980; 9:475-493.

12. Klimentzou P, Paravatou-Petsotas M, Zikos C, Beck A, Skopeliti M, Czarnecki J. Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha. Peptides 2006; 27:183-193.

13. Hashish EA, Zhang C, Ruan X, Knudsen DE, Chase CC, Isaacson RE, et al. A multi epitope fusion antigen elicits neutralizing antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea Virus in vitro. Clin Vaccine Immunol 2013; 20:1076–1083.

14. Acres SD, Babiuk LA. Studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay. J Am Vet Med Assoc 1978; 173:555-559.

15. Parreño V, Marcoppido G, Vega C, Garaicoechea L, Rodriguez D, Saif L, et al. Milk supplemented with immune colostrum: Protection against rotavirus diarrhea and modulatory effect on the systemic and mucosal antibody responses in calves experimentally challenged with bovine rotavirus. Immunol Immunopathol 2010; 136:12-27.

16. Shahbazi P, Shayan P, Ebrahimzadeh E, Rahbari S. Specific egg yolk antibody against recombinant Cryptosporidium parvum P23 protein. Iran J Parasitol 2009; 4:15-24.

17. Nikbakht G, Jahntigh M, Assadian F. Generation of egg yolk antibodies in chicken (IgY) against influenza M2 (M2e) protein. International Conference on Chemical, Biological and Medical Sciences (ICCBMS'2012). 2012 August 25-26; Kuala Lumpur (Malaysia): 2012. p. 20-22.

18. Hu Q, Zhang X, Li L, Xu F, Lv Y. Production of Egg Yolk Antibody (IgY) against recombinant Porcine Epidemic Diarrhea Virus COE protein. Acta Sci Vet 2014; 42:1181.

19. Borhani K, Mohabati-Mobarez A, Khabiri AR, Behmanesh M, Khoramabadi N. Production of specific IgY Helicobacter pylori recombinant OipA protein and assessment of its inhibitory effects towards attachment of H. pylori to AGS cell line.  Clin Exp Vaccine Res 2015; 4:177-183.

20. Hideaki Y, Peralta RC, Roger D, Sadako S, Yutaka I, Kodama Y. Passive protective effect of chicken egg Yolk immunoglobulins against experimental Enterotoxigenic Eschenichia coli infection in neonatal piglets. Infect Immun 1992; 60:998-1007.

21. Marquardt RR, Jin LZ, Kim JW, Fang L, Frohlich AA, Baidoo SK. Passive protection effect of egg yolk antibodies against Entrotoxigenic Escherichia coli K88+ infection in neonatal and early weaned piglets. FEMS Immunol Med Microbiol 1999; 23:283-288.

22. Malekan M, Moosakhani F, Pourtaghi H, Bahonar A, Malekan M. Production of immunized eggs against Salmonella enteritidis and Escherichia. coli k99. Vet Clin Path (Vet J Tabriz) 2010; 4:759-764.

23. Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR. Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine. Altern Lab Anim 2005; 33: 129-154.

24. Nasiri K, Nassiri MR, Tahmoorespur M,  Haghparast A, Zibaee S. Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen. Pol J Vet Sci 2016; 19: 271-279.

25. Liou JF, Shiau JW, Tai C, Chen LR. Production of 

egg yolk immunoglobulin against Escherichia coli from white Leghorn and Lohmann chickens. J Anim Vet Adv 2011; 10:2349-2356.

26. Yuan P, Yanshun C, Zhiqiang S, Xiaoxiang N, Jinliang W, GuangJun G, et al. Co-expressions of fimbrial subunit gene K88 and K99 of Enterotoxigenic Escherichia coli. Chin J Vet Sci 2009; 29:437-440.

27. Schade R, Behn I, Erhard M, Hlinak A, Staak C. Chicken egg yolk antibodies, production and application: IgY-Technology. Germany: Springer; 2000.

28. Yokoyama H, Peralta RC, Diaz R, Sendo S, Ikemori Y, Kodoma Y. Passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets. Infect Immun 1992; 60: 998-1007.