奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2015.04.016

›› 2015, Vol. 42 ›› Issue (4): 877-882.doi: 10.16431/j.cnki.1671-7236.2015.04.016

奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

刘云¹, 贾斌¹, 石峰², 李鑫², 李洪涛³, 张永生¹, 宁梦影¹, 纪俊明¹, 秦炳燕¹

1. 石河子大学动物科技学院, 石河子 832003;
2. 石河子大学生命科学学院, 石河子 832003;
3. 石河子大学医学院, 石河子 832003

收稿日期:2014-10-30 出版日期:2015-04-20 发布日期:2015-05-05
通讯作者: 贾斌 E-mail:jiabin@shzu.edu.cn
作者简介:刘云(1988-),女,新疆巴州人,硕士生,研究方向:基础兽医学,E-mail:lyly_ly1995123@163.com
基金资助:
兵团博士基金项目(Zfy基因干扰生产奶牛性控精液的研究(2012BB016))

Prokaryotic Expression,Purification of Early Pregnancy Factor Protein of the Dairy Cattle and Preparation of Polyclonal Antibody against it

LIU Yun¹, JIA Bin¹, SHI Feng², LI Xin², LI Hong-tao³, ZHANG Yong-sheng¹, NING Meng-ying¹, JI Jun-ming¹, QIN Bing-yan¹

1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. College of Life Science, Shihezi University, Shihezi 832003, China;
3. School of Medcine, Shihezi University, Shihezi 832003, China

Received:2014-10-30 Online:2015-04-20 Published:2015-05-05

摘要/Abstract

摘要： 本研究旨在表达、纯化奶牛早孕因子重组蛋白及制备多克隆抗体.本试验构建奶牛早孕因子重组蛋白原核表达载体pET32a-EPF,转化至大肠杆菌BL21(DE3)内进行诱导表达,SDS-PAGE切胶纯化的早孕因子重组蛋白免疫BALB/c小鼠制备多克隆抗体,并用Western blotting和ELISA对重组蛋白及抗体进行检测.结果显示,成功表达并纯化了奶牛早孕因子重组蛋白,重组蛋白分子质量约37 ku,表达量约占菌体总蛋白的48.6%,纯化后目的蛋白纯度可达93%,重组蛋白可与所制备的多克隆抗体结合,ELISA测定的抗体效价为1:12 800.结果表明,本研究成功表达并纯化了奶牛早孕因子重组蛋白,为研究奶牛早孕诊断奠定基础.

关键词: 早孕因子; 原核表达; SDS-PAGE; Western blotting; ELISA

Abstract: The study was aimed to express and purify the dairy cattle early pregnancy factor (EPF) protein and prepare EPF polyclonal antibody. The recombinant plasmid pET32a-EPF was expressed in E.coli BL21 (DE3). Polyclonal antibodies were developed by immunizing BALB/c mice with the SDS-PAGE gel extraction purified fusion protein. The recombinant protein and the polyclonal antibody were separately detected by Western blotting and ELISA. The results showed that the recombinant protein with a molecular weight of 37 ku was expressed successfully in E.coli and its content was approximately 48.6% of total bacteria proteins. The purity of target protein could reach 93% after purification. The recombinant protein was recognized by the prepared polyclonal antibody in Western blotting analysis. The antibody titer was 1:12 800.These results indicated that we had successfully obtained and purified the dairy cattle EPF protein, which laid a foundation for the further study of dairy cattle pregnancy diagnosis.

Key words: early pregnancy factor; prokaryotic expression; SDS-PAGE; Western blotting; ELISA

中图分类号:

S823

刘云, 贾斌, 石峰, 李鑫, 李洪涛, 张永生, 宁梦影, 纪俊明, 秦炳燕. 奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备[J]. , 2015, 42(4): 877-882.

LIU Yun, JIA Bin, SHI Feng, LI Xin, LI Hong-tao, ZHANG Yong-sheng, NING Meng-ying, JI Jun-ming, QIN Bing-yan. Prokaryotic Expression,Purification of Early Pregnancy Factor Protein of the Dairy Cattle and Preparation of Polyclonal Antibody against it[J]. , 2015, 42(4): 877-882.

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奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

Prokaryotic Expression,Purification of Early Pregnancy Factor Protein of the Dairy Cattle and Preparation of Polyclonal Antibody against it

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