Expression of UreB and HspA of Helicobacter pylori in silkworm pupae and identification of its immunogenicity

• Published in: Molecular biology reports Vol. 38; no. 5; pp. 3173 - 3180
• Main Authors: Zhang, Xiaolin, Shen, Weide, Lu, Ye, Zheng, Xiaojian, Xue, Renyu, Cao, Guangli, Pan, Zhonghua, Gong, Chengliang
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.06.2011; Springer Netherlands; Springer Nature B.V
• Subjects: Animal Anatomy; Animal Biochemistry; Animals; antibodies; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Biomedical and Life Sciences; Bombyx - growth & development; Bombyx - immunology; Bombyx - microbiology; Bombyx mori nucleopolyhedrovirus; Escherichia coli; Female; fluorescence; genes; green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Heat shock proteins; Heat-Shock Proteins - genetics; Heat-Shock Proteins - immunology; Helicobacter pylori; Helicobacter pylori - chemistry; Helicobacter pylori - immunology; Histology; Humans; immune response; immunization; immunoglobulin G; Life Sciences; Lymphoma; Mice; Mice, Inbred BALB C; Molecular biology; Molecular Sequence Data; Morphology; Pathogens; polyacrylamide gel electrophoresis; protein subunits; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - immunology; protein synthesis; Pupa - immunology; Pupa - microbiology; pupae; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; silkworms; Urease - chemistry; Urease - genetics; Urease - immunology; Western blotting; Protein expression; Oral vaccine; Silkworm pupa; Heat shock protein A subunit and urease B subunit of
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-010-9988-2

For mass production of urease B subunit (UreB) and heat shock protein A subunit (HspA) of Helicobacter pylori with Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) and to determine whether they could be used as an oral vaccine against H. pylori, besides, to determine the time course of expressed recombinant protein and the optimum acquisition time directly through green fluorescence, HspA and enhanced green fluorescence protein (EGFP) genes were cloned into vector pFastBacDual to form donor vector pFastBacDual-(EGFP) (HspA), UreB gene was cloned into vector pFastBacDual to form donor vector pFastBacDual-UreB,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP) (HspA) and Bacmid-UreB respectively. They were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB. Using these recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB inoculated the silkworm pupae, a recombinant HspA and UreB protein were expressed in silkworm pupae, which were around 13 and 62 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After oral immunization of mice, serum specific IgG antibodies against HspA and UreB in vaccine group were much higher than that in mock and native silkworm powder control groups. The results indicated that the expressed recombinant HspA and UreB in silkworm pupae would possess good immunogenicity. In addition, when EGFP and HspA proteins were expressed, a direct correlation between the increase in intensity of fluorescence and HspA concentration.