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猪卵巢经过冻切破碎,采用0.05M pH 8 PBS充分振荡,通过一系列孔径不同的尼龙筛,过筛后外观浓集得到大量的猪卵子。随后一组采用0.1M pH9.5硼酸钠68℃水浴孵育50分钟溶解猪卵透明带抗原(PZ);另一组将猪卵提取物用组织研磨器处理制成匀浆,通过(NH_4)_2SO_4分级沉淀提纯PZ抗原。分别经高速离心(5℃,27000g/分×20分钟)和Sephadex-G100柱层析加以分离纯化。热溶解制备的PZ和由(NH_4)_2SO_4分级沉淀提取的PZ,通过疑胶过滤各分离出二和三个蛋白质峰,经电泳鉴定,结合文献报告考虑PZ蛋白组分主要集中在第一峰。经凝胶过滤纯化的PZ热溶性蛋白组分,通过7.5％聚丙烯酰胺凝胶电泳鉴定为二条带,SDS-聚丙烯酰胺凝胶电泳测定PZ相对迁移率为0.62、0.60,分子量测定为65,000和67,000。免疫双扩散证实,纯化的PZ抗原具有免疫反应的组织特异性,并保持了免疫活性。
Porcine ovaries were crushed by freezing, shaking well with 0.05M pH 8 PBS, passing through a series of nylon sieves with different pore sizes to obtain a large number of pig eggs after screening. A subsequent group was incubated with 0.1M sodium borate pH9.5 in water bath at 68 ° C for 50 minutes to dissolve the porcine zona pellucida antigen (PZ); the other group, the porcine egg extract was treated with a tissue grinder to homogenize and passed through (NH_4) _2SO_4 PZ antigen was purified by fractional precipitation. They were separated by high speed centrifugation (5 ℃, 27000g / min × 20min) and Sephadex-G100 column chromatography. PZ prepared by thermal dissolution and PZ extracted by (NH 4) 2 SO 4 fractionation were separated by gel filtration. Two and three protein peaks were separated and identified by electrophoresis. Considering the PZ protein components mainly concentrated in the first peak in the literature, The PZ-soluble protein fractions purified by gel filtration were identified as two bands by 7.5% polyacrylamide gel electrophoresis. The relative mobility of PZ was 0.62 and 0.60 respectively by SDS-polyacrylamide gel electrophoresis and the molecular weight was 65,000 and 67,000. Immune double diffusion confirmed that the purified PZ antigen has the tissue specificity of the immune response and maintains the immunocompetence.