- Email: [email protected]
Evaluation of immunogenicity in bovines of Riphicephalus (Boophilus) microplus and Haemaphysalis longicornis recombinant calreticulins
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
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necessary to identify other tick antigens that can be used in a cocktail to induce anti-tick immunity more potently preventing the transmission of pathogens of tick-borne diseases.
Supported by CNPq, FAPERGS, FINEP, PRONEX (Brazil) and JSPS (Japan).
doi:10.1016/j.vetimm.2008.10.147
Evaluation of DNA immunization with plasmids expressing rBYC (Boophilus Yolk Pro-Cathepsin)
Evaluation of immunogenicity in bovines of Riphicephalus (Boophilus) microplus and Haemaphysalis longicornis recombinant calreticulins Luís Fernando Imamura 4 , Misao da Silva Vaz Jr. 1,2
Parizi 1,2,∗ , Herbert Rech 1,2 , Saiki 4 Onuma , Aoi Masuda 1,3 , Itabajara
1
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonc¸alves, 9500, Porto Alegre, RS 91501970, Brazil 2 Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil 3 Departamento de Biologia Molecular e Biotecnologia, UFRGS, RS, Brazil 4 Hokkaido University, Sapporo, Japan Keywords: Calreticulin; Vaccine; Riphicephalus (Boophilus) microplus e Haemaphysalis longicornis E-mail address: [email protected] (L.F. Parizi). Species: Ruminants The ticks Riphicephalus (Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasities of bovines, causing serious damages to the livestock production. The main method of control is based on the acaricides. However, the use of vaccines has been studied as a promising control method. The calreticulin (CRT) is a multifunctional protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite–host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) cloned in pET-5b and H. longicornis (rHlCRT) cloned in pET43a were expressed in Escherichia coli and purified by ion exchange chromatography and used for immunization of bovines. The fraction used for tests in vitro was purified by ion exchange and gel filtration chromatography. By ELISA, it was demonstrated that both CRTs are recognized by immunized bovines. The immunogenic and antigenic capacities of rBmCRT and rHlCRT were analyzed by two methods. In silico, despite the difference in amino acid sequences, antigenic index analysis of rHlCRT and rBmCRT with the Jameson–Wolf algorithm indicated that both proteins were very similar in the antigenicity index. Although six different regions between the tick CRTs have been determined. In vitro, this data were corroborated by competitive ELISA that suggests the presence of different epitopes between proteins. By Western blot, anti-native rBmCRT and rHlCRT bovine sera also recognized the native proteins in larvae extracts. These results demonstrate the presence of shared epitopes between recombinant and native proteins. In conclusion, the results suggest that the rBmCRT and rHlCRT could have a similar immunogenicity for bovines.
doi:10.1016/j.vetimm.2008.10.148
Maria Lúcia S. Medeiros 1,∗ , Alexandre T. Leal 1 , Carlos Logullo 5 , Sandra E. Farias 1,4 , Aoi Masuda 1,3 , Itabajara da Silva Vaz Jr. 1,2 1
C. Biotecnologia-UFRGS, Brazil Fac. Veterinária-UFRGS, Brazil 3 Depto Biol. Molecular e Biotecnologia-UFRGS, Brazil 4 Depto Fisiologia-UFRGS, Brazil 5 LQFPP-CBB-UENF, Campos dos Goytacazes, RJ, Brazil Keywords: B. microplus; Vaccine DNA; Boophilus Yolk ProCathepsin; Embryogenesis 2
E-mail address: [email protected] (M.L.S. Medeiros). Species: Ruminants The Boophilus microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the DNA immunization containing BYC cDNA could elicit the specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18-Neo). XL1-Blue E. coli were transformed with clones, BYC-pcDNA3 or BYC-pME18-Neo, and the plasmids were purified by alkaline lysis method. In order to evaluate immunogenicity of BYC, BALB/c mice were immunized with DNA vaccines by intramuscular injection. The mice received two intramuscular inoculations of 100 g plasmids DNA (BYC-pcDNA3 or BYC-pME18-Neo) and the negative controls received only PBS, pcDNA3 or pME-18Neo. The production of antibody after the immunizations was evaluated by Western Blot and ELISA. Antibodies against BYC were detected in mice inoculated with BYCpcDNA3. These results show that DNA vaccination produces specific anti-BYC antibodies and suggest that DNA could prove useful for vaccine development. Supported by FAPERGS, FINEP, PRONEX. doi:10.1016/j.vetimm.2008.10.149
279
necessary to identify other tick antigens that can be used in a cocktail to induce anti-tick immunity more potently preventing the transmission of pathogens of tick-borne diseases.
Supported by CNPq, FAPERGS, FINEP, PRONEX (Brazil) and JSPS (Japan).
doi:10.1016/j.vetimm.2008.10.147
Evaluation of DNA immunization with plasmids expressing rBYC (Boophilus Yolk Pro-Cathepsin)
Evaluation of immunogenicity in bovines of Riphicephalus (Boophilus) microplus and Haemaphysalis longicornis recombinant calreticulins Luís Fernando Imamura 4 , Misao da Silva Vaz Jr. 1,2
Parizi 1,2,∗ , Herbert Rech 1,2 , Saiki 4 Onuma , Aoi Masuda 1,3 , Itabajara
1
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonc¸alves, 9500, Porto Alegre, RS 91501970, Brazil 2 Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil 3 Departamento de Biologia Molecular e Biotecnologia, UFRGS, RS, Brazil 4 Hokkaido University, Sapporo, Japan Keywords: Calreticulin; Vaccine; Riphicephalus (Boophilus) microplus e Haemaphysalis longicornis E-mail address: [email protected] (L.F. Parizi). Species: Ruminants The ticks Riphicephalus (Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasities of bovines, causing serious damages to the livestock production. The main method of control is based on the acaricides. However, the use of vaccines has been studied as a promising control method. The calreticulin (CRT) is a multifunctional protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite–host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) cloned in pET-5b and H. longicornis (rHlCRT) cloned in pET43a were expressed in Escherichia coli and purified by ion exchange chromatography and used for immunization of bovines. The fraction used for tests in vitro was purified by ion exchange and gel filtration chromatography. By ELISA, it was demonstrated that both CRTs are recognized by immunized bovines. The immunogenic and antigenic capacities of rBmCRT and rHlCRT were analyzed by two methods. In silico, despite the difference in amino acid sequences, antigenic index analysis of rHlCRT and rBmCRT with the Jameson–Wolf algorithm indicated that both proteins were very similar in the antigenicity index. Although six different regions between the tick CRTs have been determined. In vitro, this data were corroborated by competitive ELISA that suggests the presence of different epitopes between proteins. By Western blot, anti-native rBmCRT and rHlCRT bovine sera also recognized the native proteins in larvae extracts. These results demonstrate the presence of shared epitopes between recombinant and native proteins. In conclusion, the results suggest that the rBmCRT and rHlCRT could have a similar immunogenicity for bovines.
doi:10.1016/j.vetimm.2008.10.148
Maria Lúcia S. Medeiros 1,∗ , Alexandre T. Leal 1 , Carlos Logullo 5 , Sandra E. Farias 1,4 , Aoi Masuda 1,3 , Itabajara da Silva Vaz Jr. 1,2 1
C. Biotecnologia-UFRGS, Brazil Fac. Veterinária-UFRGS, Brazil 3 Depto Biol. Molecular e Biotecnologia-UFRGS, Brazil 4 Depto Fisiologia-UFRGS, Brazil 5 LQFPP-CBB-UENF, Campos dos Goytacazes, RJ, Brazil Keywords: B. microplus; Vaccine DNA; Boophilus Yolk ProCathepsin; Embryogenesis 2
E-mail address: [email protected] (M.L.S. Medeiros). Species: Ruminants The Boophilus microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the DNA immunization containing BYC cDNA could elicit the specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18-Neo). XL1-Blue E. coli were transformed with clones, BYC-pcDNA3 or BYC-pME18-Neo, and the plasmids were purified by alkaline lysis method. In order to evaluate immunogenicity of BYC, BALB/c mice were immunized with DNA vaccines by intramuscular injection. The mice received two intramuscular inoculations of 100 g plasmids DNA (BYC-pcDNA3 or BYC-pME18-Neo) and the negative controls received only PBS, pcDNA3 or pME-18Neo. The production of antibody after the immunizations was evaluated by Western Blot and ELISA. Antibodies against BYC were detected in mice inoculated with BYCpcDNA3. These results show that DNA vaccination produces specific anti-BYC antibodies and suggest that DNA could prove useful for vaccine development. Supported by FAPERGS, FINEP, PRONEX. doi:10.1016/j.vetimm.2008.10.149