Cloning and expression of porA gene as the first step of a vaccine candidate study against Neisseria meningitidis serogroup A infection
| Autor: | P Afrough, A Behrouzi, M Davari, MA Malekan, A Fateh, F Vaziri, SD Siadat |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: | |
| Zdroj: | Vaccine Research, Vol 3, Iss 3, Pp 60-63 (2016) |
| Druh dokumentu: | article |
| ISSN: | 2383-2819 2423-4923 |
| Popis: | Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in human. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic Porin. This study aimed to clone and determine the expression of PorA as the first step for producing a proper antigen in a vaccine study against N. meningitidis. Methods: An approximately 1200-bp fragment of porA gene was amplified by PCR using N. meningitidis serogroup A genomic DNA and then cloned into prokaryotic expression vector pET-28a. The resulting construct (pET28a-porA plasmid) was transformed into competent E.coli BL21 cells for expression of recombinant protein. The proper overexpression of the recombinant protein was verified by SDS-PAGE and Western Blotting. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. The nucleotide sequence homology of the cloned porA gene was 97% , compared to the reference gene (NCBI GenBank accession number AL157959.1). The prokaryotic expression system (pET28a-porA- BL21) could produce our 45-kDa target recombinant protein, efficiently. Conclusion: The prokaryotic expression system and conditions used in this study provides an applicable method for producing recombinant PorA and possibly many other bacterial outer membrane proteins for future vaccine studies. |
| Databáze: | Directory of Open Access Journals |
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