| 小鼠卵ZP3蛋白的可溶性表达、纯化及免疫活性鉴定 |
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| 引用本文： | 孙美玉,马正海,李永鑫,吕涛,陈开旭,张富春.小鼠卵ZP3蛋白的可溶性表达、纯化及免疫活性鉴定[J].生物工程学报,2009,25(8):1166-1172. |
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| 作者姓名： | 孙美玉 马正海 李永鑫 吕涛 陈开旭 张富春 |
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| 作者单位： | 新疆大学生命科学与技术学院新疆生物资源基因工程国家重点实验室,乌鲁木齐,830046 |
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| 基金项目： | 国家自然科学基金项目(No.307600136);;教育部春晖计划(No.200603)资助~~ |
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| 摘 要： | 哺乳动物卵透明带3(Zona pellucida 3,ZP3)在诱导获能精子发生顶体反应中发挥着重要作用,其在大肠杆菌中主要以包涵体形式表达。本研究在大肠杆菌中诱导表达可溶性的mZP3融合蛋白,并鉴定该蛋白的免疫活性。将小鼠卵透明带3克隆至pMAL-p2x质粒,转化至大肠杆菌BL21表达菌中。用不同温度、不同IPTG浓度、不同诱导时间及不同浓度的添加剂诱导目的蛋白表达,以筛选出mZP3融合蛋白可溶性表达的最佳条件。大量表达纯化后以Western blotting和ELISA检测蛋白的免疫活性。酶切鉴定及DNA测序表明mZP3已克隆入pMAL-p2x质粒。经优化诱导表达条件,筛选出mZP3融合蛋白可溶性表达的最佳条件为:当A600为0.6时添加0.02 mol/L葡萄糖,以0.6 mmol/L IPTG于25oC条件下诱导表达4 h。ELISA及Western blotting检测结果表明诱导表达的蛋白具有免疫活性。在大肠杆菌中表达并纯化获得可溶性mZP3蛋白,为mZP3免疫不育疫苗研制及其免疫效果检测提供了可溶性抗原。
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| 关 键 词： | 小鼠 卵透明带3 可溶性蛋白 原核表达 |
| 收稿时间： | 2009/1/12 0:00:00 |
Soluble expression, purification and immunoreactive identification of mouse zona pellucida 3 fusion protein |
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| Meiyu Sun,Zhenghai Ma,Yongxin Li,Tao Lu,Kaixu Chen,Fuchun Zhang.Soluble expression, purification and immunoreactive identification of mouse zona pellucida 3 fusion protein[J].Chinese Journal of Biotechnology,2009,25(8):1166-1172. |
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| Authors: | Meiyu Sun Zhenghai Ma Yongxin Li Tao Lu Kaixu Chen Fuchun Zhang |
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| Institution: | Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China;Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China;Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China;Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China;Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China;Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China |
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| Abstract: | Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction.In this study,we obtained the soluble mZP3 fusion protein and identified its immunoreactivity.mZP3 cDNA was cloned into plasmid pMAL-p2x,and the recombinant plasmid was transformed into Escherichia coli BL21.To get the soluble mZP3 fusion protein,we tried to optimize the expression conditions,including additives,IPTG concentrations,temperatures and induction duration.Then,Western blotting and ELI... |
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| Keywords: | mouse zona pellucida 3 soluble protein prokaryotic expression |
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