| Objective: Screening for the ZNF185 epitope;Synthesis of four peptide antigens carrying the ZNF185 epitope,and further study the immune response and immunocontraceptive effect induced by corresponding synthetic peptides.Methods:(1)By using DNA Star software to predict the secondary structure,hydrophilicity,plasticity,surface accessibility and antigenic index of ZNF185 protein,and determine the dominant epitope of ZNF185,and further optimize and optimize the dominant antigen,prepare and purify the polypeptide.Finally,the four peptide antigens that carry the ZNF185 epitope are synthesized.(2)7-8 weeks of male mice were selected,and the mice were immunized according to the following three factors: antigen(antigen peptide-?,antigen peptide-?,antigen peptide-? and antigen peptide-?),immune dose: 80?g,immune pathways: hypodermic injection(Hypo),and negative control group(PBS)and blank control group;(3)After the mice were injected with immunization,blood was collected from the tail vein,and serum was precipitated,then the antibody titer was measured by ELISA;(4)The optimal concentration of the selected antigen peptide was screened again by ELISA;(5)Make a paraffin section,the structural changes of testis and epididymis were detected by HE staining;(6)After mice were injected with immunization,when the antibody titer reaches the peak or the antibody titer returns to normal levels,the immunized mice were subjected to 1: 2(male: female)cage to study the effects of immunological contraceptive effect and fertility recovery;(7)After mice were injected with immunization,detection of testicular and epididymal coefficient,sperm density,viability,deformity rate and other indicators of sperm quality;(8)Each experiment was repeated three times under the same conditions,using SPSS 17.0 statistical software for data analysis,Graphpad-prism 6.04 software for mapping.Results:(1)The ZNF185 epitope was successfully selected and the purity of the fusion polypeptide ZNF185 obtained by total chemical synthesis was greater than 90%;(2)ELISA results showed that the antibody titer of antigen peptide-? immunization group was significantly higher than that of other antigen peptide groups,and gradually increased with the increase of the number of immunization weeks,peaked at 12 weeks after immunization(P>0.05),and the peak value was 1: 4000,initially successfully selected to produce high antibody titer antigen peptide-?;(3)The selected antigen peptide-? was again subjected to the optimal concentration detection.The ELISA results showed that the antibody titer of the antigen peptide-? immunization experimental group was significantly higher than that of the control group(P<0.05);(4)The results of HE staining showed that there was no significant difference in the testis and epididymis tissue structure between the immunized experimental groups compared with the blank control group and the negative control group(P>0.05);the sperm density in the epididymal ducts of each immunized experimental group was significantly reduced(P<0.05);(5)After the mice were immunized,the caged experiment results showed that: 17 weeks after immunization,the antigen peptide-The mating rate and pregnancy rate of the III immunization group were significantly lower than those of the control group(P<0.05),indicating that the antigen peptide-? has more immunocontraceptive effect and the litter size per litter does not differ significantly(P>0.05);when the antibody titer When recovered to a normal level,the mating rate and pregnancy rate in the immunized experimental group all increased significantly;(6)Counts of epididymal sperm counts and viability and viability statistics showed that the antigen peptide was comparable to the blank control and negative control group.The number of sperms,sperm motility,and viability of the immunized group were significantly lower(P<0.05),which was consistent with the contraceptive effect of mice after immunization;(7)There was a significant difference in epididymal sperm abnormality in the antigenic peptide-? experimental group compared with the PBS negative control group,with statistical significance.(P<0.05).Conclusion: ZNF185 contraceptive vaccine has a certain resistance to fertility. |
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