Cloning and Expression of Mannheimia haemolytica PlpE Gene in Escherichia coli and its Immunogenicity Assessment

Document Type: Original Articles

Authors

Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

Abstract

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-β-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.

Keywords

Main Subjects


Article Title [French]

Clonage et Expression du Gène PlpE de Mannheimia haemolytica dans Escherichia coli et Évaluation de son Immunogénicité

Abstract [French]

Mannheimia haemolytica est responsable de pertes économiques considérables dans l’industrie agroalimentaire de nombreuses régions du monde. Cette bactérie est l’un des agents responsables de la fièvre des navires chez les bovins. Les vaccins actuels contre M. haemolytica sont modérément efficaces car ils ne fournissent pas une protection complète contre la maladie. La production d'un vaccin économique pour la protection des animaux d'élevage contre M. haemolytica a donc attiré l'attention de nombreux scientifiques. Les protéines de la membrane externe (OMP) jouent un rôle majeur dans la pathogenèse et l'immunogénicité de M. haemolytica. Des recherches sur les OMP de M. haemolytica ont montré que les anticorps dirigés contre les OMP, à savoir les PlpE, peuvent jouer un rôle important dans la protection immunitaire. Dans cette étude, le gène de M. haemolytica, OMP PlpE, a été cloné dans le vecteur d'expression pET26-b, puis exprimé dans Escherichia coli BL21. L'expression de la protéine a été menée par l’induction de cellules Bl21 d'Escherichia coli en culture avec 1 mM d'isopropyl-β-D-thiogalactopyranoside. La PlpE recombinante a été purifiée en utilisant une résine d'agarose Ni-NTA, puis soumise à une électrophorèse sur gel de dodécylsulfate de sodium et de polyacrylamide. L'identité de la protéine exprimée a été analysée par Western blot montrant que la protéine rPlpE était exprimée et produite correctement. Afin d'évaluer l'immunogénicité de la protéine recombinante, la rPlpE purifiée a été utilisée comme antigène pour la production d'anticorps chez les chèvres. Les résultats obtenus suggèrent que la protéine recombinante peut être utilisée comme antigène candidat pour le développement de vaccins et/ou de tests de diagnostic.

Keywords [French]

  • Mannheimia haemolytica
  • PlpE
  • Cloning
  • Expression
  • Immunogenicity
Aubry, P., Warnick, L.D., Guard, C.L., Hill, B.W., Witt, M.F., 2001. Health and performance of young dairy calves vaccinated with a modified-live Mannheimia haemolytica and Pasteurella multocida vaccine. J Am Vet Med Assoc 219, 1739-1742.

Ayalew, S., Confer, A.W., Blackwood, E.R., 2004. Characterization of immunodominant and potentially protective epitopes of Mannheimia haemolytica serotype 1 outer membrane lipoprotein PlpE. Infect Immun 72, 7265-7274.

Ayalew, S., Confer, A.W., Payton, M.E., Garrels, K.D., Shrestha, B., Ingram, K.R., et al., 2008. Mannheimia haemolytica chimeric protein vaccine composed of the major surface-exposed epitope of outer membrane lipoprotein PlpE and the neutralizing epitope of leukotoxin. Vaccine 26, 4955-4961.

Belshe, R.B., Smith, M.H., Hall, C.B., Betts, R., Hay, A.J., 1988. Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection. J Virol 62, 1508-1512.

Confer, A.W., 1993. Immunogens of Pasteurella. Vet Microbiol 37, 353-368.

Confer, A.W., Ayalew, S., Panciera, R.J., Montelongo, M., Whitworth, L.C., Hammer, J.D., 2003. Immunogenicity of recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE and augmentation of a commercial vaccine. Vaccine 21, 2821-2829.

Confer, A.W., McCraw, R.D., Durham, J.A., Morton, R.J., Panciera, R.J., 1995. Serum antibody responses of cattle to iron-regulated outer membrane proteins of Pasteurella haemolytica A1. Vet Immunol Immunop 47, 101-110.

Confer, A.W., Panciera, R.J., Fulton, R.W., Gentry, M.J., Rummage, J.A., 1985. Effect of vaccination with live or killed Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis. Am J Vet Res 46, 342-347.

Confer, A.W., Panciera, R.J., Gentry, M.J., Fulton, R.W., 1987. Immunologic response to Pasteurella haemolytica and resistance against experimental bovine pneumonic pasteurellosis, induced by bacterins in oil adjuvants. Am J Vet Res 48, 163-168.

Conlon, J.A., Shewen, P.E., Lo, R.Y., 1991. Efficacy of recombinant leukotoxin in protection against pneumonic challenge with live Pasteurella haemolytica A1. Infect Immun 59, 587-591.

Eskandari, M.H., Hosseini. A., Alasvand Zarasvand, S., Aminlari, M., 2012. Cloning, Expression, Purification and Refolding of Caprine Prochymosin. Food Biotechnol 26, 143-53.

Gerlach, G.F., Anderson, C., Klashinsky, S., Rossi-Campos, A., Potter, A.A., Willson, P.J., 1993. Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1. Infect Immun 61, 565-572.

Haag, A.F., Ostermeier, C., 2009. Positive-selection vector for direct protein expression. Biotechniques 46, 453-457.

Hodgson, J.C., Moon, G.M., Quirie, M., Donachie, W., 2003. Association of LPS chemotype of Mannheimia (Pasteurella) haemolytica A1 with disease virulence in a model of ovine pneumonic pasteurellosis. J Endotoxin Res 9, 25-32.

Morton, R.J., Panciera, R.J., Fulton, R.W., Frank, G.H., Ewing, S.A., Homer, J.T., et al., 1995. Vaccination of cattle with outer membrane protein-enriched fractions of Pasteurella haemolytica and resistance against experimental challenge exposure. Am J Vet Res 56, 875-879.

Mosier, D.A., Simons, K.R., Confer, A.W., Panciera, R.J., Clinkenbeard, K.D., 1989. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect Immun 57, 711-716.

Pandher, K., Confer, A.W., Murphy, G.L., 1998. Genetic and immunologic analyses of PlpE, a lipoprotein important in complement-mediated killing of Pasteurella haemolytica serotype 1. Infect Immun 66, 5613-5619.

Pandher, K., Murphy, G.L., Confer, A.W., 1999. Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1. Vet Microbiol 65, 215-226.

Perino, L.J., Hunsaker, B.D., … 31, 1997. A review of bovine respiratory disease vaccine field efficacy. Bovine Pr 31, 59-66.

Potter, A.A., Schryvers, A.B., Ogunnariwo, J.A., Hutchins, W.A., Lo, R.Y., Watts, T., 1999. Protective capacity of the Pasteurella haemolytica transferrin-binding proteins TbpA and TbpB in cattle. Microb Pathog 27, 197-206.

Purdy, C.W., Livingston, C.W., Jr., Frank, G.H., Cummins, J.M., Cole, N.A., Loan, R.W., 1986. A live Pasteurella haemolytica vaccine efficacy trial. J Am Vet Med Assoc 188, 589-591.

Purdy, C.W., Raleigh, R.H., Collins, J.K., Watts, J.L., Straus, D.C., 1997. Serotyping and enzyme characterization of Pasteurella haemolytica and Pasteurella multocida isolates recovered from pneumonic lungs of stressed feeder calves. Curr Microbiol 34, 244-249.

Rahn, R., Thomaidis, G., Frenkel, G., Frank, P., Kinner, U., 1989. [Late results of conservative condylar fracture treatment]. Dtsch Z Mund Kiefer Gesichtschir 13, 197-202.

Rajeev, S., Kania, S.A., Nair, R.V., McPherson, J.T., Moore, R.N., Bemis, D.A., 2001. Bordetella bronchiseptica fimbrial protein-enhanced immunogenicity of a Mannheimia haemolytica leukotoxin fragment. Vaccine 19, 4842-4850.

Rice, J.A., Carrasco-Medina, L., Hodgins, D.C., Shewen, P.E., 2007. Mannheimia haemolytica and bovine respiratory disease. Anim Health Res Rev 8, 117-128.

Shewen, P.E., Wilkie, B.N., 1988. Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can J Vet Res 52, 30-36.

Srinand, S., Hsuan, S.L., Yoo, H.S., Maheswaran, S.K., Ames, T.R., Werdin, R.E., 1996a. Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays. Vet Microbiol 49, 181-195.

Srinand, S., Maheswaran, S.K., Ames, T.R., Werdin, R.E., Hsuan, S.-L., 1996b. Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis. Vet Microbio 52, 81-89.

Studier, F.W., 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207-234.

Studier, F.W., Moffatt, B.A., 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.

Zecchinon, L., Fett, T., Desmecht, D., 2005. How Mannheimia haemolytica defeats host defence through a kiss of death mechanism. Vet Res 36, 133-156.