中文摘要：

为制备rpZP3a蛋白供发展避孕疫苗研究，将编码天然提取pZP3a上的DNA序列（446—1423）插入至毕赤酵母分泌型表达载体pPICZaA上，重组质粒pPICZaA—pZP3a线性化后通过电穿孔转入毕赤酵母GS115，经抗生素Zeoein筛选获得工程菌。在2L发酵罐中，用甲醇诱导工程菌进行高密度发酵生产rpZP3a。分离浓缩发酵上清液，通过螯合铜离子的亲和柱纯化rpZP3a，用SDS-PAGE和Westernblot进行鉴定，以Quantity One软件对rpZP3a进行定量分析并计算纯度和回收率。用rpZP3a免疫家兔，以ELISA法和间接免疫荧光法检测抗血清对rpZP3a和猪卵透明带的抗体反应。获得了分泌表达rpZP3a的工程菌，其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，命名为rpZP3a，平均产量为8mg／L，纯度达92％。回收率为63％。用其免疫家兔获得抗rpZP3a抗血清，ELISA测定显示能与rpZP3a和天然提取pZP3反应。间接免疫荧光法分析显示抗rpZP3a抗血清能与猪卵透明带反应，产生亮绿荧光。用酵母表达系统成功表达了rpZP3a，该蛋白保留有天然pZP3的免疫活性。

英文摘要：

To obtain the recombinant pZP3a protein for the study of the contraceptive vaccines,the DNA sequence（446-1423） encoding purified pZP3a was inserted into a vector pPICZaA. The recombinant plasmid pPICZaA-pZP3ct was linearized and then transformed into Pichia pastoris GSll5 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3ct in high-density fermentation. Then rpZP3a was purified by Cu^2＋ metal affinity column chromatography from the separated and concentrated fermentative supematants. The purified rpZP3a was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3a were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3a. The antibody responses against rpZP3a and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3a in secretion were constructed. A 46kD component named rpZP3a which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3a obtained from fermentative supematants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3a antiserum was prepared by immunization of a male rabbit with purified rpZP3a. This anti-rpZP3a antiserum could react with rpZP3a and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3a （46kD） protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.