Verification of the Presence of Capsule Gene Sequences in Nasopharyngeal Isolates of Nontypeable Haemophilus Influenzae From Healthy Children at a Brazilian Day Care Center

Silva, Maria Emilia Bonifácio da; Marin, José Moacir

doi:10.1590/s1517-83822001000300014

Cited by 1 publication

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“…Haemophilus influenzae has been classified into two major groups, based on the presence of polysaccharide capsule; unencapsulated (nontypeable H. influenzae ) (NTHi) and encapsulated (typeable H. influenzae ) strains, in both of which the pe gene that is responsible for the production of adhesion protein, is present ( 1 , 2 ). There are six types of encapsulated H. influenzae : a, b, c, d, e, and f ( 3 ).…”

Section: Introductionmentioning

confidence: 99%

Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

Tavakoli

Bouzari

Siadat

et al. 2015

Jundishapur J Microbiol

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Background:Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis.Objectives:In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study.Materials and Methods:At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting.Results:The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration.Conclusions:The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway.

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Section: Introductionmentioning

confidence: 99%