In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens
Background: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. Materials and Methods: The purpose of the current study was to in silico design a L7/L12-SO...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2018-01-01
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| Series: | Advanced Biomedical Research |
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| Online Access: | http://www.advbiores.net/article.asp?issn=2277-9175;year=2018;volume=7;issue=1;spage=21;epage=21;aulast=Golshani |
| _version_ | 1819098353614454784 |
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| author | Maryam Golshani Melina Ghasemian Nematollah Gheibi Saeid Bouzari |
| author_facet | Maryam Golshani Melina Ghasemian Nematollah Gheibi Saeid Bouzari |
| author_sort | Maryam Golshani |
| collection | DOAJ |
| description | Background: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. Materials and Methods: The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. Results: Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-β-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 μg/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. Conclusions: Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis. |
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| format | Article |
| id | doaj.art-65e84019480449e581945a269b7286d1 |
| institution | Directory Open Access Journal |
| issn | 2277-9175 |
| language | English |
| last_indexed | 2024-12-22T00:29:38Z |
| publishDate | 2018-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Advanced Biomedical Research |
| spelling | doaj.art-65e84019480449e581945a269b7286d12022-12-21T18:44:58ZengWolters Kluwer Medknow PublicationsAdvanced Biomedical Research2277-91752018-01-0171212110.4103/abr.abr_10_17In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b AntigensMaryam GolshaniMelina GhasemianNematollah GheibiSaeid BouzariBackground: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. Materials and Methods: The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. Results: Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-β-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 μg/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. Conclusions: Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.http://www.advbiores.net/article.asp?issn=2277-9175;year=2018;volume=7;issue=1;spage=21;epage=21;aulast=GolshaniBrucellacloningfusion proteinin silico designL7/L12outer membrane protein 2b |
| spellingShingle | Maryam Golshani Melina Ghasemian Nematollah Gheibi Saeid Bouzari In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens Advanced Biomedical Research Brucella cloning fusion protein in silico design L7/L12 outer membrane protein 2b |
| title | In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens |
| title_full | In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens |
| title_fullStr | In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens |
| title_full_unstemmed | In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens |
| title_short | In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens |
| title_sort | in silico design and in vitro expression of a fusion protein encoding brucella abortus l7 l12 and somp2b antigens |
| topic | Brucella cloning fusion protein in silico design L7/L12 outer membrane protein 2b |
| url | http://www.advbiores.net/article.asp?issn=2277-9175;year=2018;volume=7;issue=1;spage=21;epage=21;aulast=Golshani |
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