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: 

• Q78