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本实验以兔 ZPB作为免疫抗原 ,利用 DNA疫苗技术进行兔免疫避孕疫苗的研究。利用 Eco RI内切酶将 PGEM-RC5 5的兔 ZP基因 RC5 5亚克隆至 p Bluescript载体 ,再经 Hind III/ Xbal双酶切后 ,将 RC5 5基因构建至真核表达质粒载体 PCMV4,得到PCMV4-RC5 5。在 PCMV4-RC5 5 DNA疫苗免疫新西兰兔的实验中 ,设雌兔实验组及雄兔实验组 ,以 Bupivacaine-HCL为辅助剂进行大腿肌肉注射 ,2 4h后在相同部位注射 2 5 0μg PCMV4-RC5 5 DNA,并在第 4周免疫加强一次。分别于注射 PCMV4-RC5 5前及注射后第 2、4、5、6、7、8周耳静脉取血 ,收集血清 ,采用 EL ISA法检测新西兰兔产生抗 ZP-RC5 5抗体水平。检测结果表明 ,PCMV4-RC5 5 DNA疫苗能诱导雌兔和雄兔产生免疫应答 ,抗体滴度最高可达 1∶ 80 0 0 ,滴度维持在 1∶ 40 0 0水平能持续两个月以上。
In this study, rabbits ZPB as an immune antigen, the use of DNA vaccine technology rabbit immunization contraceptive vaccine research. The EcoRI endonuclease was used to subclone the rabbit ZP gene RC5 5 of PGEM-RC5 5 into the p Bluescript vector. After double digestion with Hind III / Xbal, the RC5 5 gene was constructed into the eukaryotic expression plasmid vector PCMV4 to obtain PCMV4 -RC5 5. In the experiment of immunization of New Zealand rabbits with PCMV4-RC5 5 DNA vaccine, the experimental group of the female rabbits and the experimental group of the male rabbits were intramuscularly injected with Bupivacaine-HCL as adjuvant. After 24 hours, 250 μg of PCMV4-RC5 5 DNA, and boosted at Week 4. Blood samples were taken from the veins before 2,4,5,6,7 and 8 weeks after injection of PCMV4-RC5 5, and the levels of anti-ZP-RC5 5 antibody in New Zealand rabbits were detected by ELISA. The results showed that the PCMV4-RC5 5 DNA vaccine induced immune responses in both rabbits and male rabbits. The highest antibody titer was 1:80 0 0 and the titer was maintained at 1: 40 0 0 for more than two months.