| 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。 |
| 关键词: 奶山羊 白细胞介素6 转移生长因子β1 融合蛋白 |
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| 基金项目: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） |
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| Preparation and function detection of purified fusion protein of dairy goat IL-6 and TGF-β1 |
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PANG Ming,DONG Jiayi,NI Silu,et al
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| Abstract: |
| 【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. |
| Key words: dairy goat IL-6 TGF-β1 purified fusion protein |
