Development of shark VNARs targeting Helicobacter pylori virulence factor HtrA
Objective To construct an immune VNAR phage display library,and develop potent VNARs targeting HtrA which is a critical virulence factor of Helicobacter,pylori from the library.Methods A white-spotted bamboo shark was immunized with recombinant HtrA protein,and the coding sequence of VNARs was cloned from its peripheral blood leukocytes(PBLs)and spleen tissues.VNAR phage display library was constructed,and anti-HtrA VNARs were selected from the library.The recombinant,expression and purification of VNARs were carried out by using Escherichia coli expression system.ELISA was used to detect the affinity and stability of VNARs.Results VNAR phage display library targeting HtrA was constructed,and two VNARs(G11 and 1A5)were selected,expressed in E.coli BL21(DE3)cells,and purified for further characterization.The affinity and stability of VNARs were assessed.The affinity values of Gi1 and 1A5 were 21.2,27.3 nmol/L,respectively,and they showed high stability in low pH solutions.A bivalent VNAR Bi-1A5 was constructed and showed a significant increase in antigen binding affinity.Conclusion VNAR molecules with high affinity targeting HtrA could be obtained by immunizing marine species whitespotted bamboo shark,which provided a new idea for the diagnosis and treatment of H.pylori infection.
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