华北农学报 ›› 2015, Vol. 30 ›› Issue (3): 31-36. doi: 10.7668/hbnxb.2015.03.006

所属专题: 畜牧 生物技术

• 论文 • 上一篇    下一篇

绵羊FecB基因的原核表达及其抗体制备

丁强, 张利平, 贾建磊, 王静, 张勇   

  1. 甘肃农业大学 动物科学技术学院, 甘肃 兰州 730070
  • 收稿日期:2015-03-18 出版日期:2015-06-28
  • 通讯作者: 张利平(1962-),女,甘肃陇南人,教授,博士,博士生导师,主要从事绵羊遗传育种与繁殖研究。
  • 作者简介:丁强(1989-),男,安徽滁州人,硕士,主要从事绵羊遗传育种与繁殖研究。
  • 基金资助:
    绵羊双羔性状分子调控机理研究项目(GNSW-2012~24);通过蛋白表达差异与动态组成研究绵羊双羔性状的信号分子及其调控机理项目(31260547)

Prokaryotic Expression of FecB Gene and Generation of Its Polyclonal Antibody

DING Qiang, ZHANG Li-ping, JIA Jian-lei, WANG Jing, ZHANG Yong   

  1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
  • Received:2015-03-18 Published:2015-06-28

摘要: 为了进一步研究 FecB 基因,利用PCR-RFLP技术筛选出 FecB 基因,PCR扩增 FecB 基因编码区序列1 509 bp, 利用限制性内切酶 Bam H Ⅰ和EcoR Ⅰ定向插入原核表达载体pET30a(+),构建原核表达载体pET30a(+)- FecB,诱导表达纯化重组蛋白,免疫小鼠制备多克隆抗体,Western Blot检测重组蛋白的免疫活性,ELISA检测抗体效价。结果表明,成功的构建了绵羊 FecB 基因的原核表达载体,并在大肠杆菌中表达目的蛋白,经过4次免疫后,获得多克隆抗体,ELISA方法检测小鼠抗体免疫效价达到1: 32 000,纯化的蛋白具有免疫活性。为研究绵羊 FecB 基因蛋白的功能提供参考。

关键词: FecB基因, 原核表达, 蛋白纯化, 多克隆抗体制备

Abstract: Further study on FecB gene,the complete FecB gene was amplified with PCR method using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank.And FecB gene was cloned into vector pET30a(+) for expressed in E.coli BL21(DE3).The expression of 6×His and FecB fusion protein was induced by IPTG,then purified by Ni-NTA chromatographic method.Polyclonal antibodies were prepared by immunized mice with the purified recombinant protein.Expression of the target protein was detected by SDS-PAGE,the specificity and titer of the antibody in anti-sera was determined by Western Blot and ELISA respectively.The recombinant FecB can be expressed by IPTG induction and purified by Ni-NTA resin.The results of ELISA and Western Blot proved that polyclonal antiserum prepared with purified recombinant FecB as antigen has high titration(>1: 32 000)and specificity.A method for prokaryotic expression and purification of sheep FecB was established and the anti-FecB polyclonal antibody with high titration and specificity were obtained.These results would provide reliable tools for the future study on FecB function.

Key words: FecB gene, Prokaryotic expression, Protein purification, Polyclonal antibody

中图分类号: 

引用本文

丁强, 张利平, 贾建磊, 王静, 张勇. 绵羊FecB基因的原核表达及其抗体制备[J]. 华北农学报, 2015, 30(3): 31-36. doi: 10.7668/hbnxb.2015.03.006.

DING Qiang, ZHANG Li-ping, JIA Jian-lei, WANG Jing, ZHANG Yong. Prokaryotic Expression of FecB Gene and Generation of Its Polyclonal Antibody[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(3): 31-36. doi: 10.7668/hbnxb.2015.03.006.

使用本文

0
    /   /   推荐 /   导出引用

链接本文: http://www.hbnxb.net/CN/10.7668/hbnxb.2015.03.006

               http://www.hbnxb.net/CN/Y2015/V30/I3/31