摘要
AIM: To construct a recombinant vector which can express Mr 26 000 outer membrane protein (OMP) from Helicobacter pylori (Hp), and to obtain the vaccine protecting against Hp infection and a diagnostic reagent kit quickly detecting Hp infection.METHODS: The gene encoding the structural Mr 26000 outermembrane protein of Hp was amplified from Hpchromosomal DNA by PCR, and inserted in the prokaryoticexpression vector pET32a ( + ), which was transformed intothe Topl0 E. coli strain. Recombinant vector was selected,identified and transformed into BL-21(DE3) E. coli strain.The recombinant fusion proteins were expressed. Theantigenicity of recombinant protein was studied by ELISA orimmunoblotting and immunized Balb/c mice.RESULTS: The gene of Mr 26 000 OMP was amplified to be594 base pairs, 1.1% of the cloned genes was mutated and1.51% of amino acid residues was changed, but there washomogeneity between them. The recombinant fusion proteinencoded objective polypeptides of 198 amino acid residues,corresponding to calculated molecular masses of Mr 26000.The level of soluble expression products was about 38.96 %of the total cell protein. After purification by Ni-NTA agaroseresin columniation, the purity of objective protein becameabout 90 %. The EESA results showed that recombinantfusion protein could be recognized by patient serum infectedwith Hp and rabbit serum immunized with the recombinantprotein. Furthermore, Balb/ c mice immunized with therecombinant proteln were protected against H. pyloriinfection.CONCLUSION: Mr 26 000 OMP may be a candidate vaccinepreventing Hp infection.
AIM:To construct a recombinant vector which can express M,26000 outer membrane protein(OMP)from Helicobacter pylori(Hp),and to obtain the vaccine protecting against Hp infection and a diagnostic reagent kit quickly detecting Hp infection. METHODS:The gene encoding the structural M_r 26000 outer membrane protein of Hp was amplified from Hp chromosomal DNA by PCR,and inserted in the prokaryotic expression vector pET32a(+),which was transformed into the Top10 E.coli strain.Recombinant vector was selected, identified and transformed into BL-21(DE3)E.coli strain. The recombinant fusion proteins were expressed.The antigenicity of recombinant protein was studied by ELISA or immunoblotting and immunized Balb/c mice. RESULTS:The gene of M_r 26 000 OMP was amplified to be 594 base pairs,1.1% of the cloned genes was mutated and 1.51% of amino acid residues was changed,but there was homogeneity between them.The recombinant fusion protein encoded objective polypeptides of 198 amino acid residues, corresponding to calculated molecular masses of M_r 26000. The level of soluble expression products was about 38.96 % of the total cell protein.After purification by Ni-NTA agarose resin columniation,the purity of objective protein became about 90 %.The ELISA results showed that recombinant fusion protein could be recognized by patient serum infected with Hp and rabbit serum immunized with the recombinant protein.Furthermore,Balb/ c mice immunized with the recombinant protein were protected against H.pylori infection.CONCLUSION: Mr 26 000 OMP may be a candidate vaccine preventing Hp infection.
