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:: Volume 12, Issue 4 (winter 2012) ::
J Ardabil Univ Med Sci 2012, 12(4): 408-417 Back to browse issues page
Cloning, Expression and Purification of P647-153 of Haemophilus influenzae
Farshad Nojoomi, Ashraf Mohabati Mobarez , Ali Hatef Salmanian, Seiyed Davar Siadat, Nima Khoramabadi, Haniyeh Aghababa
Abstract:   (4091 Views)

  Background & Objectives: Haemophilus influenzae is a Gram-negative bacterium that is part of the normal nasopharyngeal flora of most humans. H. influenzae strains were defined in two categories: encapsulated (typeable) and non-capsulated (nontypeable) strains. Outer membrane protein P6 is a highly conserved and stable protein in the outer membrane of both encapsulated and non-capsulated strains of H. influenzae. As an immunogen, P6 protein is a potential candidate vaccine against H. influenzae strains. The aim of this study was to produce recombinant protein P6 as a carrier protein for production of conjugate vaccines.

  Methods: The sequence (324 bp) coding P647-153 protein of H. influenzae was successfully cloned in pJET1.2 and subsequently in pET28a (+). Expression of the recombinant protein was induced with 1mM IPTG. Recombinant P647-153 was purified through dissolving inclusions in 8M urea buffer, absorbing to Ni-NTA resins, washing by buffers with decreasing urea concentration and finally eluted in Imidazole solution. Imidazole was removed by dialysis against PBS (pH 7.4). The recombinant p6 47-153 was confirmed by western blot analysis using rabbit anti H. influenzae polyclonal antiserum.

  Results: The recombinant P647-153 was successfully expressed in E. coli BL21 (DE3) and purified (4 mg /lit of broth culture). The immunoblotting showed that recombinant P647-153 conserved its native antigenic structure.

  Conclusion: Western blot results, along with that of sequencing, confirmed accurate production of recombinant P647-153 and partially retention of its conformational epitopes.

Keywords: Haemophilus influenzae; P647-153; Cloning and Expression
Full-Text [PDF 363 kb]   (2771 Downloads)    
Type of Study: article | Subject: Special
Received: 2011/10/7 | Accepted: 2012/01/30 | Published: 2012/12/21
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Nojoomi F, Mohabati Mobarez A, Salmanian A H, Siadat S D, Khoramabadi N, Aghababa H. Cloning, Expression and Purification of P647-153 of Haemophilus influenzae. J Ardabil Univ Med Sci. 2012; 12 (4) :408-417
URL: http://jarums.arums.ac.ir/article-1-77-en.html


Volume 12, Issue 4 (winter 2012) Back to browse issues page
مجله دانشگاه علوم پزشکی اردبیل Journal of Ardabil University of Medical Sciences
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