猪白细胞介素2／6嵌合基因的融合表达及活性

采用重叠延伸PCR（SOE—PcR）方法通过一基因柔性接头（linker）将猪白介素2（PoIL-2）、6（PoIL-6）基因构建成PoIL-2linker-PoIL-6嵌合基因并克隆入pQE-30原核表达载体中进行融合表达；对表达的重组融合蛋白（rPoIL-2qinker-PoIL-6）进行纯化。分别检测rPoIL-2-linker—PoIL-6蛋白与抗PoIL-2、PoIL-6单抗发生特异性免疫反应及促猪外周血淋巴细胞和猪脾脏细胞的增殖活性。结果显示：成功构建了PoIL-2-linker-PoIL-6嵌合基因及其重组原核表达质粒（rpQE-30／PoIL-2-linker-PoIL-6）。表达的rPoIL-2linker-PoIL-6蛋白相对分子质量约28000，蛋白经纯化后纯度在96％以上。rPoIL-2-linker—PoIL-6蛋白分别具有与单一重组PoIb2、PoIL-6蛋白（rPoIL-2、rPoIL-6）对照相近的生物学活性，可与抗PoIL-2、PoIL-6单抗发生特异性免疫反应，并可显著促进猪外周血淋巴细胞和猪脾脏细胞的增殖。结果表明，rPoIL-2-linker—PoIL-6蛋白在体外具有单一rPoIL-2和rPoIL-6蛋白的双重生物学活性，这为下一步进行rPoIL-2-linker-PoIL-6蛋白在动物体内活性研究奠定了基础。

Expression and bioactivities of porcine interleukin-2/6 fusion protein in vivo and in vitro

In order to explore a high efficient porcine genetic engineering immune enhancement agent for vaccine adjuvant,the recombinant chimeric gene of PoIL-2-linker-PoIL-6 constructed by porcine interleukin-2(PoIL-2) gene linked interleukin-6(PoIL-6) gene via a flexible linker by splicing overlap extension PCR(SOE-PCR) method was cloned into prokaryotic expression vector pQE30.The recombinant PoIL-2-linker-PoIL-6 fusion protein(rPoIL-2-linker-PoIL-6) was expressed in a pQE30/E.coli JM109 system and purified with the innovated recombinant fusion protein purification method.The activities of rPoIL-2-linker-PoIL-6 protein were estimated through detecting its specific immune response to monoclonal antibody(MAb) against PoIL-2 and PoIL6 by ELISA assay,and the abilities of promoting the proliferation of porcine peripheral blood T lymphocyte(PBLC) and spleen lymphoblast cells.The results indicated chimeric gene of PoIL-2-linker-PoIL-6 was successfully constructed and abundantly fusion expressed in E.coli.,and the fusion rPoIL-2-linker-PoIL-6 protein was successfully purified with the molecular mass of about 28 000 and more than 95% pure on SDS-PAGE.The rPoIL-2-linker-PoIL-6 protein displayed the specific immune response to monoclonal antibody(MAb) against PoIL-2 and PoIL-6,as well as the bioactivities of significant promoting the proliferation of porcine PBLC and spleen lymphoblast cells in vivo.This study primarily proved that rPoIL-2-linker-PoIL-6 protein had the duplex bioactivity of PoIL-2 and PoIL-6 protein in vitro,which laid a foundation for further widely use of rPoIL-2 and PoIL-6 protein in vivo.