| 人IL-3基因原核表达载体的构建及表达 |
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| 引用本文： | 严学倩,尹郸丹,付伟,韩骅,梁英民.人IL-3基因原核表达载体的构建及表达[J].现代生物医学进展,2011,11(15):2832-2835. |
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| 作者姓名： | 严学倩 尹郸丹 付伟 韩骅 梁英民 |
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| 作者单位： | 第四军医大学唐都医院血液内科第四军医大学遗传与发育生物学教研室,陕西西安,710038 |
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| 基金项目： | 国家科技重大专项，国家自然科学基金面上项目，国家自然科学基金 |
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| 摘 要： | 目的:构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法:通过佛波酯(TPA)和植物球血凝素(PHA)刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应(RT-PCR)获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a(+)中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果:酶切鉴定和测序结果证明成功构建了原核表达载体pET32a-IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论:成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。
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| 关 键 词： | 白细胞介素3 造血干细胞 原核表达 |
Construction and Expression of the hIL-3-Prokaryotic Expression Vector |
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| YAN Xue-qian,YIN Dan-dan,FU Wei,HAN Hua,LIANG Ying-min.Construction and Expression of the hIL-3-Prokaryotic Expression Vector[J].Progress in Modern Biomedicine,2011,11(15):2832-2835. |
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| Authors: | YAN Xue-qian YIN Dan-dan FU Wei HAN Hua LIANG Ying-min |
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| Institution: | △(Department of Hematology,Tangdu Hospital,Department of Medical Genetics and Developmental Biology, Fourth Military Medical University,Xi’an 710038,China) |
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| Abstract: | Objective:To construct the prokaryotic expression vector of human IL-3 pET32a-IL-3,and to expresse the prutein in Escherichia coli.Methods:The human T cell leukemia cell line Jurkat cells were stimulated by phorbol ester(TPA) and phytohaemagglutinin (PHA) to increase the expression of IL-3mRNA.The cDNA was got by using the RT-PCR.The human IL -3 gene was amplified by polymerase chain reaction(PCR) with the cDNA of Jurkat cells as the template.The amplified fragment was cloned into the pET32a(+) vector,and t... |
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| Keywords: | Interleukin-3 Hematopoietic stem cells Prokaryotic expression |
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