title: Expression of UreB and HspA of Helicobacter pylori in silkworm pupae and identification of its immunogenicity.
abstract: 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.
authors.affiliation: School of Pre-clinical Medicine and Biological Science, Medical College, Soochow University, 215123 Suzhou, People's Republic of China. zhangxiaolin8@126.com
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhang
authors.forename: Weide
authors.initials: W
authors.lastname: Shen
authors.forename: Ye
authors.initials: Y
authors.lastname: Lu
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Zheng
authors.forename: Renyu
authors.initials: R
authors.lastname: Xue
authors.forename: Guangli
authors.initials: G
authors.lastname: Cao
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Pan
authors.forename: Chengliang
authors.initials: C
authors.lastname: Gong
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D012831:Bombyx / Q000254:growth & development / Q000276:immunology / Q000382:microbiology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D016480:Helicobacter pylori / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011679:Pupa / Q000276:immunology / Q000382:microbiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014510:Urease / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014510:Urease