摘要
为研制具有鸡白细胞介素-6、2(ChIL-6,ChIL-2)协同免疫增强作用的鸡基因工程复合免疫增强剂,采用重叠延伸PCR(SOE-PCR)方法将ChIL-6和ChIL-2基因构建成ChIL-6-linker-ChIL-2嵌合基因;对嵌合基因进行原核表达,采用自行创新的专利纯化方法对表达的rChIL-6-linker-ChIL-2蛋白(重组融合蛋白)进行纯化。采用间接ELISA方法检测重组融合蛋白分别与抗ChIL-6、抗ChIL-2单克隆抗体的特异性免疫反应活性,采用MTS法检测重组融合蛋白体外促鸡外周血T淋巴细胞和脾淋巴细胞的增殖活性。结果显示,成功构建了ChIL-6-linker-ChIL-2嵌合基因及其重组表达质粒rpQE-30-ChIL-6-linker-ChIL-2,表达的重组融合蛋白的分子质量约为40ku,纯化后的纯度在96%以上。该重组融合蛋白可分别与抗ChIL-6、抗ChIL-2单克隆抗体发生特异性免疫反应,并具有显著的促鸡外周血T淋巴细胞和脾淋巴细胞增殖活性,其活性优于任一重组蛋白对照。本研究结果初步证明了rChIL-6-linker-ChIL-2蛋白在对鸡免疫细胞的增殖作用方面与rChIL-6和rChIL-2蛋白的作用相似,为进一步研究rChIL-6-linker-ChIL-2蛋白在鸡体内的活性奠定了基础。
To develop a highly efficient chicken complex genetic engineering immune enhancement agent for vaccine adjuvant with the synergy bioactivities of chicken interleukin-6and 2(ChIL-6and ChIL-2),the recombinant chimeric gene of ChIL-6-linker-ChIL-2was constructed by ChIL-2gene linked with ChIL-6via a flexible linker by splicing overlap extension PCR(SOE-PCR)method.The chimeric gene was then cloned into prokaryotic expression vector pQE-30.The recombinant ChIL-6-linker-ChIL-2fusion protein(rChIL-6-linker-ChIL-2)was expressed and purified with the innovated and patented recombinant fusion protein purification method.ELISA was used for testing the immune activities of rChIL-6-linker-ChIL-2protein to monoclonal antibody against ChIL-6and ChIL-2,and the abilities of the fusion protein to promote the proliferation of chicken peripheral blood T lymphocyte(PBLC)and spleen lymphoblast cells was measured by MTS method.The results indicated that the chimeric gene of ChIL-6-linker-ChIL-2and its recombinant expression plasmid(rpQE30-ChIL-6-linker-ChIL-2)were successfully constructed and the fusion rChIL-6-linker-ChIL-2protein which is 40 ku in molecular weight was successfully purified.The purity of the protein was more than 96% with SDS-PAGE analysis.The rChIL-6-linker-ChIL-2protein had the specific immune response to the monoclonal antibodies against ChIL-6and ChIL-2,and the fusion protein was significantly promoted the proliferation of chicken spleen lymphoblast cells and PBLCin vitro,which was significantly higher than either rChIL-6or rChIL-2protein control group.The results showed that rChIL-6-linker-ChIL-2protein had the similar effect with rChIL-6or rChIL-2protein on the proliferation of immune cells in vitro,which laid the foundation for the further widely study on the bioactivities of rChIL-6-linker-ChIL-2protein in vivo.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第9期974-980,共7页
Chinese Veterinary Science
基金
河南省科技创新人才计划项目(豫科人组[2011]1号)
