[Expression and biological activity identification of recombinant Hap protein of NTHi].
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.
Yao
Feng
F
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. biologyyao@shou.com
Li
Wan-yi
WY
Kuang
Yu
Y
Li
Ming-yuan
MY
Feng
Feng
F
Feng
Wei
W
Zhang
Qiang
Q
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*
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