重组人透明带蛋白3的高密度发酵表达及食蟹猴的免疫研究
发布时间：2018-12-08 16:28
【摘要】：卵透明带(zona pellucida，ZP)，是哺乳动物卵子外包裹的一层半透明的糖蛋白基质，是精子与卵细胞识别和结合的部位，同时对卵子起保护作用。卵透明带的生理特性极具研究价值，，ZP蛋白的特性使之具有开发成避孕疫苗的潜力，然而，ZP是极具组织特异性的蛋白，天然存在量很少。为了得到大量高纯度的ZP3蛋白进行深入的抗生育疫苗和免疫不孕诊断的研究，我们重组表达了人ZP3成熟肽片段。 本研究克隆了hZP3成熟肽(第23～383位氨基酸序列)的基因片段，运用基因工程方法将其重组到穿梭型载体质粒pPICZaA上，选择测序正确的重组质粒，在大肠杆菌中扩增质粒、Sac Ⅰ线性化后使用电击转化的方法把重组基因转入巴斯德毕赤酵母(Pichia pastoris)，用高浓度的Zoecin筛选到整合了高拷贝重组基因的酵母菌株，通过SDS-PAGE和Western Blotting方法鉴定甲醇诱导表达的重组蛋白，借助发酵罐进行高密度发酵表达rhZP3，目的蛋白利用载体质粒上设计的6个组氨酸纯化标记，以Ni~(2+)螯合柱亲和层析法纯化，纯化后的rhZP3使用胞壁酰二肽(Muramyldipeptide，MDP)佐剂免疫灵长类动物食蟹猴(Macaca fascicularis)，以猪ZP3包被抗原用ELISA检测抗血清，并用小鼠卵巢冰冻切片与人卵巢石蜡切片进行免疫组化鉴定，用猴抗rhZP3抗血清进行体外抗小鼠精卵结合实验以评价抗血清的抑制受精能力。 结果表明，在载体质粒中成功克隆了hZP3成熟肽的cDNA序列，用重组质粒转化并筛选得到可以稳定表达rhZP3的重组酵母，Western Blotting鉴定重组蛋白具有免疫学活性，通过发酵罐高密度表达后纯化得到高纯度的rhZP3，以之免疫食蟹猴，获得了较高滴度的抗hZP3抗体，抗体与猪ZP3有交叉反应性，猴抗rhZP3抗血清可以与小鼠和人卵母细胞上的透明带发生免疫反应，并在体外明显能抑制小鼠卵子结合精子。 研究结果说明，重组hZP3的确可以在酵母中分泌表达，并具有与天然蛋白相似的活性；抗rhZP3抗血清在体外体现了封闭透明带、阻断受精的活性。重组蛋白可以满足开发抗生育疫苗和免疫不孕诊断的需求。
[Abstract]:The pellucida zone (zona pellucida,ZP) is a translucent glycoprotein matrix encapsulated in mammalian eggs. It is the site of sperm recognition and binding with egg cells and plays a protective role on eggs. The physiological characteristics of the pellucida zone are of great value, and the ZP protein has the potential to be developed into a contraceptive vaccine. However, ZP is a highly tissue-specific protein, which is naturally present in a small amount. In order to obtain a large amount of high purity ZP3 protein for further study of antifertility vaccine and immunological infertility diagnosis, we expressed a mature peptide fragment of human ZP3. In this study, we cloned the gene fragment of mature peptide of hZP3 (23 ~ 383 amino acid sequence), recombined it into shuttle vector plasmid pPICZaA by genetic engineering method, selected the correct sequencing recombinant plasmid, and amplified the plasmid in Escherichia coli. The recombinant gene was transformed into Pichia pastoris (Pichia pastoris), by electroporation after linearization of Sac 鈪
本文编号：2368646
[Abstract]:The pellucida zone (zona pellucida,ZP) is a translucent glycoprotein matrix encapsulated in mammalian eggs. It is the site of sperm recognition and binding with egg cells and plays a protective role on eggs. The physiological characteristics of the pellucida zone are of great value, and the ZP protein has the potential to be developed into a contraceptive vaccine. However, ZP is a highly tissue-specific protein, which is naturally present in a small amount. In order to obtain a large amount of high purity ZP3 protein for further study of antifertility vaccine and immunological infertility diagnosis, we expressed a mature peptide fragment of human ZP3. In this study, we cloned the gene fragment of mature peptide of hZP3 (23 ~ 383 amino acid sequence), recombined it into shuttle vector plasmid pPICZaA by genetic engineering method, selected the correct sequencing recombinant plasmid, and amplified the plasmid in Escherichia coli. The recombinant gene was transformed into Pichia pastoris (Pichia pastoris), by electroporation after linearization of Sac 鈪
本文编号：2368646
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