重组鼠IL-28的原核表达、纯化及多克隆抗体的制备

Prokaryotic expression,purification of recombinant mouse interleukin-28 and production of its polyclonal antibody

目的：获取重组鼠IL-28（m IL-28）纯化蛋白,制备多克隆抗体。方法：用PCR技术扩增鼠IL-28成熟蛋白的编码序列,克隆入原核表达载体pET-30,构建融合表达载体pET-30 a-m IL-28,转化大肠埃希菌BL21（DE3）,以IPTG诱导表达IL-28融合蛋白,经镍柱亲和层析纯化,然后免疫6-8周龄鸡,制备多克隆抗体,采用ELISA检测抗体效价。结果：成功构建了表达载体pET-30 a-m IL-28,DNA序列测定结果与预期结果一致。在37℃培养条件下,IPTG诱导表达的IL-28融合蛋白进行SDS-PAGE电泳分析,发现其与融合蛋白的理论计算值一致,免疫鸡后收获抗血清,ELISA显示抗体效价具有高度特异性。结论：获得了重组鼠IL-28纯化蛋白,制备了鼠IL-28多克隆抗体,为进一步深入研究IL-28的生物活性及其应用打下基础。

Objective： To obtain purified recombinant interleukin-28 of mouse（mIL-28） and prepare its polyclonal antibody.Methods： The cDNA fragment coding for mature mIL-28 protein was amplified by PCR and cloned into vector pET-30 to construct fusion expression vector pET-30a-mIL-28.After pET-30a-mIL-28 was transformed into E.coli BL21（DE3）,the bacteria were induced by IPTG.The expressed mIL-28 fusion protein was purified by Ni-NTA affinity chromatography.Six to eight weeks old chickens were immunized with the purified protein for obtaining the antiserum.The titers of antibodies were measured by ELISA. Results： The DNA sequencing showed that the expression vector pET-30a-mIL-28 was constructed successfully.After induced by IPTG,the expressed mIL-28 fusion protein in E.coli cultured at 37℃ appeared a single band on SDS-PAGE.The result of ELISA indicated that the prepared polyclonal antibody had high titer and specificity. Conclusion： The purified recombinant interleukin-28 of mouse and polyclonal antibody have been acquired.