奶山羊IL-6与TGF-β1融合蛋白的制备及其活性检测  

Preparation and function detection of purified fusion protein of dairy goat IL-6 and TGF-β1

作　　者：庞明 董佳易 倪思璐 管雄 张煜琛 陈德坤[1] PANG Ming;DONG Jiayi;NI Silu;GUAN Xiong;ZHANG Yuchen;CHEN Dekun(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China;Shaanxi Xianyang Food and Drugs Inspection and Testing Center,Xianyang,Shaanxi 712000,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]陕西省咸阳市食品药品检验检测中心,陕西咸阳712000

出　　处：《西北农林科技大学学报（自然科学版）》2019年第11期1-7,15,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：青海省重点研发与转化计划项目(NMSY-2018-07);咸阳市重大科技专项(2017K01-34);陕西省重点产业创新链项目(2018ZDCXL-NY-01-06)

摘　　要：【目的】制备具有免疫活性的奶山羊白细胞介素6(Interleukin-6,IL-6)与转移生长因子-β1(Transfer growth factor-β1,TGF-β1)融合蛋白。【方法】采集奶山羊外周血,分离外周血单个核细胞(Peripheral blood mononuclear cell,PBMCs),将PBMCs用刀豆蛋白A(ConA)刺激后提取其总RNA,RT-PCR扩增IL-6与TGF-β1基因,构建其克隆载体及原核表达载体pET-32a-TGF-β1和pET-32a-IL-6,然后进行PCR和测序鉴定。将原核表达载体pET-32a-TGF-β1和pET-32a-IL-6转化至大肠杆菌BL21(DE3)中,用IPTG诱导表达,以镍柱纯化试剂盒纯化IL-6与TGF-β1重组蛋白,对表达产物和纯化后的蛋白进行SDS-PAGE。用纯化的IL-6、TGF-β1蛋白刺激PBMCs,以甘油醛-3-磷酸脱氢酶GAPDH基因为内参,采用qRT-PCR法检测PBMCs IL-17 mRNA的表达量。【结果】RT-PCR扩增获得了627 bp的IL-6基因片段和1 137 bp的TGF-β1基因片段,成功构建了IL-6和TGF-β1基因的克隆载体及pET-32a-TGF-β1和pET-32a-IL-6原核表达载体,并在大肠杆菌BL21(DE3)中得到成功表达;获得了纯化的奶山羊IL-6与TGF-β1融合蛋白,该融合蛋白联合刺激能使PBMCs的IL-17 mRNA表达水平显著升高。【结论】获得了具有免疫活性的奶山羊IL-6与TGF-β1重组蛋白。【Objective】This study aimed to obtain immunologically active purified fusion protein of dairy goat interleukin-6(IL-6)and transfer growth factor-β1(TGF-β1).【Method】Peripheral blood of dairy goats was collected and peripheral blood mononuclear cells(PBMCs)were isolated.PBMCs were stimulated by concanavalin A(ConA)and total RNA was extracted.The IL-6 and TGF-β1 genes were amplified by RT-PCR,and the cloning vector and prokaryotic expression vectors pET-32a-TGF-β1 and pET-32a-IL-6 were constructed and identified by PCR and sequencing.The prokaryotic expression vectors pET-32a-TGF-β1 and pET-32a-IL-6 were transformed into E.coli BL21(DE3)and induced by IPTG.The protein of IL-6 and TGF-β1 was purified by nickel column purification kit.The expression product and the purified protein were identified by SDS-PAGE.PBMCs were stimulated with purified IL-6 and TGF-β1 protein.The expression of IL-17 mRNA in PBMCs was detected by qRT-PCR with glyceraldehyde-3-phosphate dehydrogenase GAPDH gene as internal reference.【Result】A 627 bp IL-6 gene fragment and a 1 137 bp TGF-β1 gene fragment were obtained by RT-PCR.The cloning vectors of IL-6 and TGF-β1 genes and pET-32a-TGF-β1 and pET-32a-IL-6 were successfully constructed.Prokaryotic expression vector was successfully expressed in E.coli BL21(DE3),and purified dairy goat IL-6 and TGF-β1 fusion protein was obtained.The IL-6 and TGF-β1 fusion protein combined stimulation promoted the IL-17 mRNA expression level of PBMCs significantly.【Conclusion】The immunologically active purified fusion protein of dairy goat IL-6 and TGF-β1 was successfully obtained.

关 键 词：奶山羊 白细胞介素6 转移生长因子-Β1 融合蛋白 

分 类 号：S858.275.3[农业科学—临床兽医学]