重组猪卵透明带-3α抗原及其抗体的制备  

Preparations of Recombinant Porcine Zona Pellucida Glycoprotein-3α(rpZP3α) and Anti-rpZP3α Antibody

作　　者：高彦茹[1] 潘善培[1] 谢琪璇[1] 肖銮娟[1] 柯琼[1] 

机构地区：[1]暨南大学生殖免疫研究所,广东广州510632

出　　处：《生物技术》2006年第4期18-20,共3页Biotechnology

基　　金：国家自然科学基金资助项目("卵透明带DNA避孕疫苗诱发生殖道黏膜免疫反应的研究";30570683"重组人卵透明带ZP3蛋白生物活性的研究";30370541)

摘　　要：目的:制备重组猪卵透明带-3α(rpZP3α)抗原及其抗体供发展避孕疫苗研究。方法:在2L发酵罐中接种毕赤酵母GS115-pZP3α工程菌,高密度发酵表达rpZP3α蛋白,表达的蛋白经螯合镍离子的亲和柱纯化后,用SDS-PAGE和Western blot进行鉴定,以Quantity One软件对rpZP3α进行定量分析。rpZP3α免疫家兔,间接免疫荧光法检测抗血清对rpZP3α和猪卵透明带的抗体反应。IVF试验体外检测抗rpZP3α抗体对猪、小鼠精卵结合的影响作用。结果:工程菌高密度发酵产物经分离纯化后获得能与抗pZP3抗体反应的46kD成分,平均产量为8mg/L,用其免疫家兔获得抗rpZP3α抗血清,间接免疫荧光法分析显示此抗血清能与猪卵透明带反应,产生亮绿荧光。体外精卵结合试验中,随着兔抗rpZP3α抗血清浓度的加大,其对猪、鼠精卵结合的抑制作用也增强。ELISA结果显示抗rpZP3α抗体与rpZP3α蛋白以及天然pZP3蛋白两种抗原反应的强度和趋势基本一致。结论:通过酵母体系制备的rpZP3α及其抗体具有免疫学活性。Objective：To obtain immunogenic rpZP3 protein and anti- rpZP3α antibody for contraceptive vaccine study.lVIethods： A steady engi- ncering Pichia pastor/s GS115 - pZP3α strain designed by our group was inoculated to express rpZP3α in high- density fermentation. Then rpZP3α was purified by Ni^2＋ metal affinity column chromatography from the separated and concentrated fermentative supematants. The purified rpZP3α was identified by SDS- PAGE and Western blot, and the quantity was analyzed by Quantity One software. One male rabbit was immunized with the purified rpZP3α. The antibody responses against rtRSP3α and porcine ZP were detected by ELISA and the indirect immunofluorescence. The effect on porcine and mouse sperm- egg binding of the anti- rpZP3α antibody was assayed in vitro fertilization tests.Results：A 46kD component named rpZP3α was obtained from the concentrated fermentation supematants after purified by Ni^2＋- NTA column, and the average yield of fermentation was to 8mg/L. Analysis of indirect immunofluorescence showed that anti- rpZP3α antiserum could react with porcine zona pellucida and prodnce the bright green fluorescence. In vitro fertilization tests, the inhibiting effects on porcine/mouse sperm- egg binding were increasing with the antiserum concentration. And the reaction of antiserum with rpZP3α was almost identical with natural pZP3. Conclusion： The rpZP3α expressed in the Plchia pastoris expression system and anti - rpZP3α antibedy had immunological activity.

关 键 词：重组猪卵透明带-3α抗原 毕赤酵母 抗体 抑制作用 

分 类 号：Q786[生物学—分子生物学] R392[医药卫生—免疫学]