【摘要】 鼠透明带 3(mZP3)作为精子的初级受体 ,是鼠透明带中的一种主要糖蛋白 ,抗鼠透明带 3抗体能够阻断精卵的结合 ,达到不育的效果 ,因此mZP3是免疫避孕研究的重要候选抗原。从小鼠卵巢中提取总RNA ,分离mRNA ,反转录获得cDNA ,将cDNA连接到测序载体pUCm -T质粒上 ,通过序列测定和分析得到正确的mZP3cDNA。经内切酶EcoRI和XhoI处理 ,将mZP3cDNA克隆至融合蛋白表达载体pGEX - 4T - 1中 ,在T4DNA连接酶的作用下构建出融合表达载体pGEX -mZP3,转化大肠杆菌BL - 2 1菌株 ,利用IPTG诱导后获得可溶性的蛋白质产物 ,经过SDS -PAGE鉴定 ,表达的融合蛋白GST -mZP3分子质量约为 72kD左右 ,纯化的融合蛋白免疫兔子 ,获得效价为1∶1 0 0 0的抗血清 ,Westernblot检测抗血清具有针对mZP3融合蛋白的专一性 ,为进一步开展mZP3的免疫功能检测的研究奠定了基础更多还原

【Abstract】 The ovarian murine zona pellucida 3 glycoprotein (mZP3) is the primary sperm receptor of mZP. An antibodies raised against mZP3 can block sperm-egg interaction. Therefore, ZP3 has been considered as a candidate immunogen in the development of contraceptive vaccines. In this study, We have cloned the mZP3 cDNA derived from the mouse ovary via RT-PCR into pUCm-T vector, subsequently subcloned into a prokaryotic expression vector pGEX-4T-1, an IPTG inducible vector. This constructed pGEX-mZP3 produces a 72 kD fusion protein identified by SDS-PAGE after IPTG induction overnight culture. This fusion protein was purified by a GST affinity column and used to immunized animal to produce high titer antisera. ELISA and Western blot analysis have determined the antisera raised against the mZP3. These have laid a basis for further determination of mZP3 function and the role of vaccine development.更多还原