hPH-20 DNA疫苗对雌性小鼠生育的影响

Effect of hPH-20 DNA vaccine on fertility of female mouse

目的:观察pcDNA3.1(+)-hPH-20基因疫苗经基因枪介导肌肉接种后在小鼠体内的表达及对小鼠受孕率的影响。方法:以小鼠为实验材料,将实验组经后腿股内侧肌以基因枪多点注射重组质粒pcDNA3.1(+)-hPH-205μg,空载体对照组注射pcDNA3.1(+)5μg,空白对照组注射生理盐水200μl,于接种后取股内侧肌检测hPH-20 mRNA和蛋白的表达。采集抗血清做人精子穿去透明带仓鼠卵细胞实验,动物合笼观察受孕情况,持续4个月后进行基因疫苗的安全性检测。结果:注射部位肌肉检测到目的条带;原位杂交在肌细胞膜、胞质和部分肌细胞核可见到蓝紫色颗粒;免疫组织化学显色在肌细胞膜和胞质可见到棕褐色颗粒;实验组小鼠受孕率和对照组相比下降77.78%,同时用鼠抗hPH-20血清能明显降低精子穿卵率;外源性基因没有整合至宿主染色体。结论:hPH-20基因疫苗经基因枪介导肌肉接种有一定的免疫避孕效果。

Objective：To observe the expression of gene vaccine of pcDNA3. 1（＋）-hPH-20 and its effect to the pregnant rate in the mouse medicated by gene gun. Methods：5 μg pcDNA3.1 （＋） --hPH20 was injected into the vastus medialis of the experimental mice by gene gun, and 5μg pcDNA3.1（＋） vector and 200μl physiological saline were injected into the control groups. The expression of mRNA and protein of hPH-20 was detected after immunization through the vastus medialis. Anti- serum was collected and human sperm zona-free hamster ovum penetration assay （SPA） was performed to examine the preg- nancy. The safety of gene vaccine was detected 4 months later. Results： （1） The target band was detected in the injected muscle. （2） The bluish-violet colored granules were located in the cell membrane, cytoplasm and part of the nucleolus using in situ hybridization. （3） The brown colored granules were located in the cell membrane and cytoplasm in immunohistochemistry. （4） The pregnancy rate in the experimental group dropped by 77.78G compared with that in the controls, and mouse antiserum reduced the sperm penetration rate significantly. （5） The target band was not found by the genome DNA PCR. Conclusion：The gene vaccine of hPH 20 has immunocontraception effect through skeletal muscle medicated by gene gun.