本研究采用RT-PCR方法自细菌脂多糖(LPS)刺激的猪脾脏细胞总RNA中扩增、克隆猪白介素-6 (porcine interleukin-6,PoIL-6)成熟肽基因,并亚克隆入pQE30载体进行原核表达,对表达的重组融合猪白介素-6 (rPoIL-6)蛋白通过尿素变性、低浓度复性液复性、PBS透析等步骤进行复性、纯化,采用PoIL-6 ELISA试剂盒检测rPoIL-6蛋白与抗PoIL-6单抗发生特异性免疫反应的活性；采用MTS法检测rPoIL-6蛋白促猪脾脏细胞的增殖活性.结果表明,成功克隆了全长555 bp的PoIL-6成熟肽基因；表达的rPoIL-6蛋白分子质量大小约20 ku;纯化后的rPoIL-6蛋白纯度在95％以上,可和抗PoIL-6单抗发生特异性免疫反应,并且具有显著促猪脾脏细胞增殖活性.%The porcine interleukin-6 (PoIL-6) mature peptide gene was amplified from the total cellular RNA of porcine spleen cells induced by bacterial lipopolysaccharides (LPS) by RT-PCR. And the PCR production was subsequently cloned, sequenced and sub-cloned into the prokaryotic expression vector pQE30. The PoIL-6 mature peptide gene was expressed in E. Coli JM109 and the expressed fusion recombinant protein (rPoIL-6) was purified under the innovated recombinant fusion protein purification method of denaturing by 8 mol/L urea, refolding by a self-innovative renaturation buffer and dialyzing by PBS buffer etc. The porcine IL-6 ELISA assay was used to detect the specifically immunological activity of the rPoIL-6 protein, and the cell proliferation activity induced by rPoIL-6 protein was assessed by MTS assay. The result showed that the PoIL-6 mature peptide gene with a length of 555 bp was successfully cloned, expressed, and purified with the molecular mass of about 20 ku and more than 95% pure on SDS-PAGE, which indicated the correct PoIL-6 fusion protein had been obtained. The rPoIL-6 protein could specifically react with McAb aginast rPoIL-6 and significantly promote the proliferation of porcine spleen lymphoblast cells.
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