犬卵透明带3 DNA疫苗在小鼠模型中的避孕效果评价

Evaluation of Contraceptive Effects of Canine Zona Pellucida 3 DNA Vaccine in a Mouse Model

为了评价犬卵透明带3(CZP3)DNA疫苗的避孕效果,采用6只雌性小鼠Mus musculus肌肉注射50μg pcDNA3-CZP3质粒后30 V电压刺激6次的方法建立实验组,以6只同等注射pcDNA3质粒的雌性小鼠为阴性对照组;初免后每周采血并用ELISA方法检测小鼠的CZP3抗体水平及第6周抗体滴度;第6周与雄鼠合笼,计算产仔数;第24周收集卵巢制成HE染色切片进行观察;最后用小鼠血清进行小鼠及犬卵透明带的间接免疫荧光实验。结果显示,1)实验组小鼠从初免第2周就产生了抗CZP3的抗体,并且与阴性对照组之间的差异有统计学意义(P<0.05);第24周实验组的抗体水平与阴性对照组之间的差异有高度统计学意义(P<0.01)。2)第6周血清中的抗体1∶4000倍稀释时,实验组与阴性对照组之间的差异有高度统计学意义(P<0.01)。3)小鼠生育率由阴性对照组的100%降低至实验组的50%,平均产仔数由阴性对照组的(6.667±0.422)只降至实验组的(2.500±1.455)只,且实验组的抗体水平与产仔数呈高度负相关。4)卵巢HE染色切片结果显示,CZP3 DNA疫苗避孕的免疫未对小鼠卵巢发育产生影响。5)间接免疫荧光实验显示,实验组小鼠产生的血清可以与小鼠及犬卵子的卵透明带基质结合。综上所述,CZP3 DNA疫苗在小鼠模型中能够显著降低小鼠的生育率及产仔数,但不影响小鼠的卵巢发育。

To evaluate the contraceptive effects of the canine zona pellucida 3(CZP3)DNA vaccine,initial evaluation of the contraceptive efficacy was made in mouse model in this study.Twelve female mice were divided into 2 groups which were intramuscularly immunized with 50μg of pcDNA3-CZP3(experiment group)and pcDNA3 plasmids(negative control group),respectively,and 6 electric pulses(30 V)were delivered subsequently.After the first immunization,blood was collected weekly;the antibody levels of CZP3 and antibody titers of CZP3 at the 6th week after the first immunization were detected by ELISA.The mice were mated with male mice at the 6th week after the first immunization,the litter size of each mouse was calculated and the data were analyzed statistically.Ovaries were collected at the 24th week after the first immunization and conducted for HE staining.Finally,the indirect immunofluorescence of mouse and dog zona pellucida were detected by mouse serum.The results indicated that compared with the negative control group:1)The levels of antibodies against CZP3 were significantly increased at the 2nd week after the first immunization in the experiment group,and lasted for up to 24 weeks.2)The antibody titer of CZP3 at 1∶4000 dilution was significantly different.3)The fertility rate of mice in the experiment group fell from 100%to 50%,and the litter size fell from 6.667±0.422 to 2.500±1.455.However,the correlation between the litter size and antibody levels in the experiment group was negative.4)HE staining of ovarian sections showed that the immunization with CZP3 DNA vaccine would not influence the development of mouse ovaries.5)The results of indirect immunofluorescence assay showed that the serum of mouse immunized with CZP3 could react with mouse and dog zona pellucida.Therefore,these results suggested that CZP3 DNA vaccine could reduce the fertility rate and litter size significantly in mouse model without influencing the development of ovaries.