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

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Archives of Razi Institute
Volume 74, Issue 2
June 2019
Pages 111-118

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