[Construction, expression and antigenic study of bivalent vaccine candidate with 26,000 OMP and heat short protein A of human Helicobacter pylori].
OBJECTIVE
To construct a recombinant vector containing gene encoding heat shock protein A (HspA) and outer membrane protein (OMP) with relative molecule mass (Mr) of 13,000 and 26,000 respectively from human Helicobacter pylori (Hp) and be expressed in E. coli BL21, as well as analyse its antigenic for the exploiting vaccine of Hp.


METHODS
The target gene encoding heat shock protein A was amplified from Hp chromosome by PCR. And then digested by restricted endonuclease enzyme of kpnI, BamH I simultaneously, and inserted into the prokaryotic expression vector pET32a(+) digested by corresponding restricted endonuclease enzyme. The recombinant vector was used to select and transform for sequence analysis. After pET32a(+)/HspA and pET32a(+)/Omp(26) digested by restricted endonuclease enzyme of Hind III, BamH I simultaneously, the pET32a(+)/HspA and 26,000 OMP were taken out of agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp(26) was used to select and transform, meanwhile expressed in E. coli BL21(DE3). The antigenic of recombinant fusion protein was analysed by western blotting.


RESULTS
Enzyme digestion analysis and sequencing showed that the target genes was found to be 951 base pairs, and had been inserted into recombinant vector, but as compared with gene reported by GenBank, 1.15% of the gene mutation and 1.26% of amino acid residues change in Hp happened respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E. coli BL21, its relative molecule mass of expressed product was 59 x 10(3), while Mr of protein expressed by pET32a(+) of them was about 20 x 10(3), and soluble expression product accounted for 19.96% of total bacterial protein. After purification with Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The western blot result showed that recombinant fusion protein could be recognized by anti-Hp positive serum and monoclonal antibody of 26,000 OMP, suggesting that this protein had good antigenic.


CONCLUSIONS
The genes coding HspA and OMP with Mr 13,000 and 26,000 respectively are cloned and expressed successfully. The results obtained lay the foundation for research on development of Hp protein and DNA vaccine and a quickly diagnostic kit applying to detection of Hp infection.
Jiang
Zheng
Z
Department of Gastroenterology, the First Affiliated Hospital, Institute of Viral Hepatitis, Chongging Medical University, Chongging 400016, China.
Pu
Dan
D
Huang
Ai-long
AL
Tao
Xiao-hong
XH
Wang
Pi-long
PL
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins; D001428:Bacterial Vaccines / Q000276:immunology*; D015153:Blotting, Western; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic