The Expression And The Immunogenic Study Of The Recombinant Human Zona Pellucida ZP3 Peptides

Objective:To express the recombinant hZP3 peptides in Pichia pastoris yeast and E. coli Bacterium and to study the immunogenicity of the recombinant hZP3 peptides.Methods:The hZP3 DNA fragment was amplified by PCR and was inserted into an expression vector pGEB for the expression in E. coli. Then, the recombinant plasmid pGEBhZ3.2 were transformed to the E.coli Rosetta-gami(DE3)plys. The transformant was induced by IPTG for the expression of the recombinant hZP3 peptide. The recombinant plasmid pPICZa-hZ3.1 transformed Pichia pastoris strains were genotyped by PCR. The recombinant peptide expression was induced by methanol. The expressed peptides were analyzed by SDS-PAGE and western blotting.The recombinant hZP3.2 peptide expressed in Rosetta bacterium was separated by SDS-PAGE. Then the strip of gel corresponding to the specific band of the recombinant hZP3.2 peptide was cut for the immunization of KUNG mice by intramuscular injection. The antibody response of the sera of the immunized mice was monitored by ELISA. And the specific reaction of antiserum with natural human zona pellucida was detected by immunohistochemical assay.Results:SDS-PAGE and Western blotting showed that a specific fusion protein of approximate 33kDa was detected by anti-His tag antibody in the lysate of pPICZa-hZ3.1 transformed Pichia pastoris. The result of PCR, restriction endonuclease digestion and sequencing analysis showed that prokaryotic expression vector pGEBhZ3.2 was successfully constructed and a specific fusion protein of approximate 27kDa was detected by anti-His tag antibody in the lysate of pGEBhZ3.2 transformed Rosetta bacteria.The Antibody response against with 6×His tag was detected by ELISA and Western blotting in the antiserum of the male KUNG mice immunized with the specific fusion protein expressed by E. coli. And the immunohistochemistry showed that the antiserum specifically reacted with natural human zona pellucida.Conclusions:The engineered yeast strain carrying hZP3 gene expressed the recombinant hZP3 peptide at a low level of expression; the N-Terminal peptide of hZP3 was produced successfully in vitro, and the recombinant hZP3 peptide was immunogenic.