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Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis
Veterinary Microbiology 136 (2009) 408–410
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis
A R T I C L E I N F O
Keywords: Haemophilus parasuis OmpA gene Molecular cloning Expression
Haemophilus parasuis is the causative agent of Gla¨ssers disease, a serious problem in the pig industry all over the world (Olvera et al., 2006). Fifteen serovars of H. parasuis have been defined, but some strains are non-typable by serotyping (Rapp-Gabrielson and Gabrielson, 1992; Turni and Blackall, 2005). It is difficult to develop universal serological diagnostic tools and effective vaccines, due to this serovar diversity and the lack of cross-reaction between serovars (Oliveira and Pijoan, 2004). The outer membrane proteins (OMPs) of H. parasuis may contribute to immunity and virulence (Miniats et al., 1991; Ruiz et al., 2001). Tadjine et al. (2004) reported a 35 kDa major outer membrane protein (MOMP) of H. parasuis. The N-terminal 12 amino acid residues of this protein are Ala-Pro-Gln-Ala-Asp-Ser-Phe-Tyr-Val-GlyAla-Lys. The N-terminal homology analysis showed that this protein is related to the OmpA family of Gramnegative bacteria. The monoclonal antibody against this 35 kDa MOMP reacts with H. parasuis strains of serovar 1– 15 and also with field strains (Tadjine et al., 2004). It is believed that this protein may be a good candidate for an effective diagnostic tool for H. parasuis infection. However, the gene encoding the 35 kDa MOMP has not been characterized so far. To clone the complete open reading frame (ORF) of the gene encoding the 35 kDa MOMP, we designed a pair of degenerate primers (forward 50 -ATGAAAAAAACAKYARTYRCATTR-30 ; reverse 50 -TTACATWGTWAYKTBTTTWGW0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.11.019
RCCTTG-30 ) based on the first 12 amino acids of its Nterminal sequence (Tadjine et al., 2004). DNA from a serovar 4 (SW124) and a serovar 5 strain (Nagasaki) was used as the template for the PCR. After PCR amplification and sequencing, we successfully obtained the complete ORF encoding this protein. The ORF is 1104 bp in length and encodes a 39.5 kDa protein of 367 amino acid residues. The first 21 N-terminal residues of the derived amino acid sequence are predicted to be the signal peptide. The cleavage of the signal sequence results in a mature protein of 346 amino acids. This would have a predicted molecular mass of 37.48 kDa, which is very close to the 35 kDa MOMP of H. parasuis determined by SDS-PAGE (Tadjine et al., 2004). The mature protein consists of an OmpA-like transmembrane domain (Nterminal residues 1–205) and a sequence with the characteristics of the OmpA family (residues 226–323). A BLAST search in GenBank indicated that the amino acid sequences deduced from the genes we obtained share 54.2–74.8% homology with ompA of several Pasteurellaceae members. Therefore, we believe that the genes obtained in this study could be the ompA gene encoding the OmpA protein of H. parasuis. The sequences were submitted to the GenBank under accession nos. EU846096 and EU846097, respectively. In February 2008, Phillips et al. submitted the genomic sequence of H. parasuis 297550, in which a named gene of ompP5 (accession no. ABKM 01000058) shares a very high homology with the ompA gene that we cloned. Hence we believe that the ompP5 is the ompA gene encoding the OmpA protein of H. parasuis. The phylogenetic analysis of the ompA gene of H. parasuis and its homologous from Pasteurellaceae family is shown in Fig. 1. To express ompA of H. parasuis, the gene was inserted into an expression vector pET32 (a+). The recombinant OmpA was expressed in E. coli Rosetta (DE3). SDS-PAGE showed that a polypeptide with a molecular mass of about 59 kDa was produced (Fig. 2). To identify the recombinant OmpA, Western blotting using antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5 was performed. As shown in Fig. 3, the recombinant OmpA from serovar 4 and 5 reacts with the corresponding antiserum. Furthermore, the recombinant OmpA of serovar 4 and 5 cross-react with each other’s antiserum. This indicates that the recombinant OmpA protein has a good antigenicity and cross-reactivity between serovar 4 and 5.
Letter to the Editor / Veterinary Microbiology 136 (2009) 408–410
409
Fig. 1. The phylogenetic tree analysis of ompA gene from H. parasuis with its homologous genes from Pasteurellaceae family. The phylogenetic tree obtained using the neighbor-joining method based on the nucleotide sequence of ompA genes from H. parasuis and of ompA gene from other family Pasteurellaceae members, including Bibersteinia trehalosi, Mannheimia glucosida, Actinobacillus suis, Mannheimia haemolytica, Pasteurella haemolytica, Haemophilus ducreyi, Pasteurella multocida and Actinobacillus actinomucetemcomitans. Numbers at nodes represent bootstrap values.
The identification of the major antigens of H. parasuis is of great importance in understanding the immunogenic characterization and pathogenesis. Moreover, it will facilitate the development of universal serodiagnostic tools and effective vaccines. Therefore, we believe that the sequences of ompA from H. parasuis obtained in this study will provide useful information for further studies on the functions of OmpA of H. parasuis. Acknowledgements
Fig. 2. Expression of ompA genes from H. parasuis serovar 4 and 5 in E. coli. The amplified ompA genes of serovar 4 and 5 cloned into the pET32 (a+) plasmids which were employed to transform E. coli strain Rosetta (DE3) (lanes 2 and 3). Control E. coli cell transformed without the insert (lane 1). All of strains were grown for 2 h in the presence (+) or in the absence () of 1 mM IPTG added. The total cell proteins from each bacterial strain were separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows indicated the recombined OmpAs expressed in E. coil. M: protein molecular weight marker.
The work was supported by program ‘‘National 11th Five-year Plan Scientific and Technical Supporting Program’’ from Ministry of Science & Technology of China (Contract no. 2006BAD06A11) and program ‘‘Standardization and Sharing of Veterinary Microorganism Resource’’ from Ministry of Science & Technology of China (Contract no. 2005DKA21205-11).
Fig. 3. Western blotting analysis of recombined OmpAs of H. parasuis serovar 4 and 5. The proteins from whole cells were separated by SDS-PAGE, transferred to PVDF membrane, followed by incubation with antiserum prepared with whole bacteria of H. parasuis serovar 4 (A) or 5 (B), respectively. Lane 1: control E. coli cell transformed without the insert; lane 2: recombined OmpA of serovar 4; lane 3: recombined OmpA of serovar 5; M: Easysee western marker. All of strains were grown for 2 h in the presence (+) or in the absence () of 1 mM IPTG added.
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Letter to the Editor / Veterinary Microbiology 136 (2009) 408–410
References Miniats, O.P., Smart, N.L., Rosendal, S., 1991. Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs. Can. J. Vet. Res. 55, 37–41. Olvera, A., Cerda`-Cue´llar, M., Aragon, V., 2006. Study of the population structure of Haemophilus parasuis by multilocus sequence typing. Microbiology 152, 3683–3690. Oliveira, S., Pijoan, C., 2004. New trends on diagnosis epidemiology and control. Vet. Microbiol. 99, 1–12. Rapp-Gabrielson, V.J., Gabrielson, D.A., 1992. Prevalence of Haemophilus parasuis serovars among isolates from swine. Am. J. Vet. Res. 53, 659– 664. Ruiz, A., Oliveira, S., Torremorell, M., Pijoan, C., 2001. Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites. J. Clin. Microbiol. 39, 1757–1762. Tadjine, M., Mittal, K.R., Bourdon, S., Gottschalk, M., 2004. Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protection role in mice. Microbiology 150, 3935–3945.
Turni, C., Blackall, P.J., 2005. Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis. Vet. Microbiol. 106, 145–151.
Bin Zhang Cheng Tang Fa-long Yang Hua Yue* College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China *Corresponding author. Tel.: +86 28 85528276; fax: +86 28 85522855 E-mail address: [email protected] (H. Yue) 11 November 2008
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis
A R T I C L E I N F O
Keywords: Haemophilus parasuis OmpA gene Molecular cloning Expression
Haemophilus parasuis is the causative agent of Gla¨ssers disease, a serious problem in the pig industry all over the world (Olvera et al., 2006). Fifteen serovars of H. parasuis have been defined, but some strains are non-typable by serotyping (Rapp-Gabrielson and Gabrielson, 1992; Turni and Blackall, 2005). It is difficult to develop universal serological diagnostic tools and effective vaccines, due to this serovar diversity and the lack of cross-reaction between serovars (Oliveira and Pijoan, 2004). The outer membrane proteins (OMPs) of H. parasuis may contribute to immunity and virulence (Miniats et al., 1991; Ruiz et al., 2001). Tadjine et al. (2004) reported a 35 kDa major outer membrane protein (MOMP) of H. parasuis. The N-terminal 12 amino acid residues of this protein are Ala-Pro-Gln-Ala-Asp-Ser-Phe-Tyr-Val-GlyAla-Lys. The N-terminal homology analysis showed that this protein is related to the OmpA family of Gramnegative bacteria. The monoclonal antibody against this 35 kDa MOMP reacts with H. parasuis strains of serovar 1– 15 and also with field strains (Tadjine et al., 2004). It is believed that this protein may be a good candidate for an effective diagnostic tool for H. parasuis infection. However, the gene encoding the 35 kDa MOMP has not been characterized so far. To clone the complete open reading frame (ORF) of the gene encoding the 35 kDa MOMP, we designed a pair of degenerate primers (forward 50 -ATGAAAAAAACAKYARTYRCATTR-30 ; reverse 50 -TTACATWGTWAYKTBTTTWGW0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.11.019
RCCTTG-30 ) based on the first 12 amino acids of its Nterminal sequence (Tadjine et al., 2004). DNA from a serovar 4 (SW124) and a serovar 5 strain (Nagasaki) was used as the template for the PCR. After PCR amplification and sequencing, we successfully obtained the complete ORF encoding this protein. The ORF is 1104 bp in length and encodes a 39.5 kDa protein of 367 amino acid residues. The first 21 N-terminal residues of the derived amino acid sequence are predicted to be the signal peptide. The cleavage of the signal sequence results in a mature protein of 346 amino acids. This would have a predicted molecular mass of 37.48 kDa, which is very close to the 35 kDa MOMP of H. parasuis determined by SDS-PAGE (Tadjine et al., 2004). The mature protein consists of an OmpA-like transmembrane domain (Nterminal residues 1–205) and a sequence with the characteristics of the OmpA family (residues 226–323). A BLAST search in GenBank indicated that the amino acid sequences deduced from the genes we obtained share 54.2–74.8% homology with ompA of several Pasteurellaceae members. Therefore, we believe that the genes obtained in this study could be the ompA gene encoding the OmpA protein of H. parasuis. The sequences were submitted to the GenBank under accession nos. EU846096 and EU846097, respectively. In February 2008, Phillips et al. submitted the genomic sequence of H. parasuis 297550, in which a named gene of ompP5 (accession no. ABKM 01000058) shares a very high homology with the ompA gene that we cloned. Hence we believe that the ompP5 is the ompA gene encoding the OmpA protein of H. parasuis. The phylogenetic analysis of the ompA gene of H. parasuis and its homologous from Pasteurellaceae family is shown in Fig. 1. To express ompA of H. parasuis, the gene was inserted into an expression vector pET32 (a+). The recombinant OmpA was expressed in E. coli Rosetta (DE3). SDS-PAGE showed that a polypeptide with a molecular mass of about 59 kDa was produced (Fig. 2). To identify the recombinant OmpA, Western blotting using antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5 was performed. As shown in Fig. 3, the recombinant OmpA from serovar 4 and 5 reacts with the corresponding antiserum. Furthermore, the recombinant OmpA of serovar 4 and 5 cross-react with each other’s antiserum. This indicates that the recombinant OmpA protein has a good antigenicity and cross-reactivity between serovar 4 and 5.
Letter to the Editor / Veterinary Microbiology 136 (2009) 408–410
409
Fig. 1. The phylogenetic tree analysis of ompA gene from H. parasuis with its homologous genes from Pasteurellaceae family. The phylogenetic tree obtained using the neighbor-joining method based on the nucleotide sequence of ompA genes from H. parasuis and of ompA gene from other family Pasteurellaceae members, including Bibersteinia trehalosi, Mannheimia glucosida, Actinobacillus suis, Mannheimia haemolytica, Pasteurella haemolytica, Haemophilus ducreyi, Pasteurella multocida and Actinobacillus actinomucetemcomitans. Numbers at nodes represent bootstrap values.
The identification of the major antigens of H. parasuis is of great importance in understanding the immunogenic characterization and pathogenesis. Moreover, it will facilitate the development of universal serodiagnostic tools and effective vaccines. Therefore, we believe that the sequences of ompA from H. parasuis obtained in this study will provide useful information for further studies on the functions of OmpA of H. parasuis. Acknowledgements
Fig. 2. Expression of ompA genes from H. parasuis serovar 4 and 5 in E. coli. The amplified ompA genes of serovar 4 and 5 cloned into the pET32 (a+) plasmids which were employed to transform E. coli strain Rosetta (DE3) (lanes 2 and 3). Control E. coli cell transformed without the insert (lane 1). All of strains were grown for 2 h in the presence (+) or in the absence () of 1 mM IPTG added. The total cell proteins from each bacterial strain were separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows indicated the recombined OmpAs expressed in E. coil. M: protein molecular weight marker.
The work was supported by program ‘‘National 11th Five-year Plan Scientific and Technical Supporting Program’’ from Ministry of Science & Technology of China (Contract no. 2006BAD06A11) and program ‘‘Standardization and Sharing of Veterinary Microorganism Resource’’ from Ministry of Science & Technology of China (Contract no. 2005DKA21205-11).
Fig. 3. Western blotting analysis of recombined OmpAs of H. parasuis serovar 4 and 5. The proteins from whole cells were separated by SDS-PAGE, transferred to PVDF membrane, followed by incubation with antiserum prepared with whole bacteria of H. parasuis serovar 4 (A) or 5 (B), respectively. Lane 1: control E. coli cell transformed without the insert; lane 2: recombined OmpA of serovar 4; lane 3: recombined OmpA of serovar 5; M: Easysee western marker. All of strains were grown for 2 h in the presence (+) or in the absence () of 1 mM IPTG added.
410
Letter to the Editor / Veterinary Microbiology 136 (2009) 408–410
References Miniats, O.P., Smart, N.L., Rosendal, S., 1991. Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs. Can. J. Vet. Res. 55, 37–41. Olvera, A., Cerda`-Cue´llar, M., Aragon, V., 2006. Study of the population structure of Haemophilus parasuis by multilocus sequence typing. Microbiology 152, 3683–3690. Oliveira, S., Pijoan, C., 2004. New trends on diagnosis epidemiology and control. Vet. Microbiol. 99, 1–12. Rapp-Gabrielson, V.J., Gabrielson, D.A., 1992. Prevalence of Haemophilus parasuis serovars among isolates from swine. Am. J. Vet. Res. 53, 659– 664. Ruiz, A., Oliveira, S., Torremorell, M., Pijoan, C., 2001. Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites. J. Clin. Microbiol. 39, 1757–1762. Tadjine, M., Mittal, K.R., Bourdon, S., Gottschalk, M., 2004. Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protection role in mice. Microbiology 150, 3935–3945.
Turni, C., Blackall, P.J., 2005. Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis. Vet. Microbiol. 106, 145–151.
Bin Zhang Cheng Tang Fa-long Yang Hua Yue* College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China *Corresponding author. Tel.: +86 28 85528276; fax: +86 28 85522855 E-mail address: [email protected] (H. Yue) 11 November 2008