title: [Expression and biological activity identification of recombinant Hap protein of NTHi].
abstract: OBJECTIVE
  To express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.
  
  
  METHODS
  Hap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  SDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).
  
  
  CONCLUSIONS
  Highly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. biologyyao@shou.com
authors.forename: Feng
authors.initials: F
authors.lastname: Yao
authors.forename: Wan-yi
authors.initials: WY
authors.lastname: Li
authors.forename: Yu
authors.initials: Y
authors.lastname: Kuang
authors.forename: Ming-yuan
authors.initials: MY
authors.lastname: Li
authors.forename: Feng
authors.initials: F
authors.lastname: Feng
authors.forename: Wei
authors.initials: W
authors.lastname: Feng
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D000268:Adhesiveness; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D002772:Cholera Toxin / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006192:Haemophilus Infections / Q000517:prevention & control; D006193:Haemophilus influenzae / Q000378:metabolism*; D007114:Immunization; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D002772:Cholera Toxin; D012697:Serine Endopeptidases