Expression Of Human ZP3 Chimeric Peptide In Insect Cells And Studies Of Its Activity

It is proved that natural B-cell epitope peptides on the fragment 334-341 and 341-360 of human ZP3 induce adequate antibody response without pathogenic T-cell response. hZP3 chimeric peptide(hCP), which was designed to combine the human ZP3 B-cell epitope, the fragment 334-364 of hZP3 with the helper T-cell epitope peptide from Plasmodium falciparum, will be expected to be an appropriate anti-ZP contraceptive vaccine. The gene encoding the human ZP3 B-cell epitope chimeric peptide was synthesized and succeeded to construct recombinant baculovirus AcNPV-OCC--hCP. The object of this study is to identify the expression of hCP in the Hi5 insect cells infected by recombinant baculovirus, to select expression condition, and to evaluate the immimoactivity of hCP and the ability of binding spermatozoa by the use of rabbit anti-rhZP3 antibody.Hi5 insect cells were infected with recombinant baculovirus and the transcript of hCP in insect cells was detected by RT-PCR. The supernatant was analyzed to identify the interesting protein by Tricine-SDS-PAGE; the Hi5 cells were infected with different dosage of recombinant baculovirus to result in different duration of infection; and then the optimal expression time was selected when there was high concentrative expressing product; antiserum was gained from the rabbits immuned with production expressed by recombinant rhZP3- yeast(Pichia pastoris X-33), and the product expressed by control rhZP3-yeast was used as sorbent to eliminate the non-specific antibody; the antiserum was used in immunohistochemistry of ovarian section and Western blot to detect the specificity of anti-rhZP3 antibody and the expression of hCP in insect cells and its reactivity; ELISA assay was processed to detect the inhibiting rate of rhZP3 antigen-antibody reaction with different concentrative hCP protein; Sperm samples from normal men were co-incubated with the recombined virus-transfected medium and evaluated the attachment of sperm to hCP.Results show that there are visible infected symptom and the mRNA of hCP canbe detected in the infected Hi5 cells by RT-PCR; Rabbit after immunization with production expressed by recombinant rhZP3-yeast can produce specific anti- rhZP3 antibody, though a low liter. Zona pellucida(ZP) of women ovary was positive in immunohistochemistry assay, Electrophoresis and Western blot were used to determine the hCP expressed in the medium of recombined virus-infected cells and a specfic band , which shown to have weak antigenic reactivity to the anti-rhZP3 antibody , was found in approximately 5.8KD in the both ways. The highest expression of hCP was discovered in the medium on the 6th day after infection with recombined virus. The immunoreaction of rhZP3 was inhibited when the concentration of expressed protein including hCP was above 50 g/mL in indirect competitive ELISA ,and the slope of regression equation of inhibiting rate is 0.0918. Positive responses were observed at the equator in sperm immunocytochemistry assay after co-incubating with the medium of recombined virus-infected cells.Results stated above show that hCP gene was successfully expressed and the optimal duration of recombinant baculovirus infected hi5 insect cells is 5-6 days ; The anti-rhZP3 antisrum could react with natural human ZP. A weak immunoreactivity of hCP and anti-rhZP3 antisrum is observed. Whether the sperm equator is the binding site of hCP needs further research.