Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli.
OBJECTIVE
To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose-binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.


METHODS
Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis. Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera.


RESULTS
E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG. The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supernatant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture. The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.


CONCLUSIONS
Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.
Kang
Qiao-Zhen
QZ
Department of Bioengineering, Zhengzhou University, Zhengzhou 450052, Henan Province, China.
Duan
Guang-Cai
GC
Fan
Qing-Tang
QT
Xi
Yuan-Lin
YL
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D058619:Maltose-Binding Proteins; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007106:Immune Sera; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; C092412:neutrophil-activating protein A, Helicobacter pylori