作　　者： ; ; ; ; ;

机构地区： 甘肃农业大学动物科学技术学院

出　　处： 《华北农学报》 2015年第3期31-36,共6页

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1509bp,利用限制性内切酶BamHⅠ和EcoRⅠ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,WesternBlot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∶32000,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。 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 Gen Bank. And FecB gene was cloned into vector p ET30a（ ＋） for expressed in E. coli BL21（ DE3）. The expression of 6 × His and Fec B 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 Fec B 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 Fec B as antigen has high titration（ 1 ∶ 32 000） and specificity. A method for prokaryotic expression and purification of sheep Fec B was established and the anti-Fec B polyclonal antibody with high titration and specificity were obtained. These results would provide reliable tools for the future study on Fec B function.

关 键 词： 基因 原核表达 蛋白纯化 多克隆抗体制备

领　　域： [生物学]