| 五种重组人源IL-18突变子表达质粒的构建及其在BxPC-3细胞中的表达 |
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| 引用本文： | 孙金权,许健,沈雯,吴晓莉,牟一平,张婷,沃兴德.五种重组人源IL-18突变子表达质粒的构建及其在BxPC-3细胞中的表达[J].科技通报,2012,28(3):68-74,83. |
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| 作者姓名： | 孙金权 许健 沈雯 吴晓莉 牟一平 张婷 沃兴德 |
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| 作者单位： | 1. 浙江中医药大学生命科学学院,杭州,310053
2. 浙江大学医学院附属邵逸夫医院,杭州,310029 |
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| 基金项目： | 浙江省自然科学基金(Y2080889,Y2080506);博士点基金(20090101110121);浙江省教育厅项目(Y200805242);浙江中医药大学校级课题一般项目(17108015) |
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| 摘 要： | 目的:设计获得5段hIL-18异构体,定向插入pEGFP-C1质粒形成融合基因,转染胰腺癌Bx-PC-3细胞并表达,为进一步研究改建的hIL-18蛋白功能奠定实验基础.方法:设计引物从pGEM-T-hIL-18质粒中PCR扩增得到5段不同的hIL-18片段,分别连接入pEGFP-C1载体构建不同的突变子(Mu0、Mu1、Mu2、Mu3和Mu4),转染原位胰腺癌BxPC-3细胞,以荧光显微镜观察绿色荧光蛋白的表达,RT-PCR法检测转染细胞中hIL-18表达水平.结果:(1)五种重组质粒经酶切与测序证实构建成功；(2)BxPC-3细胞转染重组质粒后可观察到绿色荧光蛋白的表达.结论:(1)成功构建了hIL-18五种异构体的绿色荧光蛋白表达载体；(2)改建的hIL-18蛋白可与绿色荧光蛋白在BxPC-3细胞融合表达.
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| 关 键 词： | pEGFP-C1 hIL- 18 BxPC-3细胞 突变子 |
Construction and Identification of Five Mutants of hIL-18 in Expression Plasmid and Their Expression in BxPC-3 Cells |
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| SUN Jinquan
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XU Jian
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SHEN Wen
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WU Xiaoli
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MOU Yiping
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ZHANG Ting
,
WO Xingde.Construction and Identification of Five Mutants of hIL-18 in Expression Plasmid and Their Expression in BxPC-3 Cells[J].Bulletin of Science and Technology,2012,28(3):68-74,83. |
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| Authors: | SUN Jinquan XU Jian SHEN Wen WU Xiaoli MOU Yiping ZHANG Ting WO Xingde |
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| Institution: | 1(1.Life Science College,Zhejiang Chinese Medical University,Hangzhou 310053,China; 2.SIR RUN RUN SHAW HOSPITAL,Affiliated with School of Medicine Zhejiang University,Hangzhou 310029,China;) |
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| Abstract: | Objective: To construct five pEGFP-C1- hIL-18 recombinant plasmids,containing five different hIL-18 isomers respectively.Method: Five different hIL-18 isomers were obtained from pGEM-T-hIL-18 plasmid by PCR and inserted into the pEGFP-C1 vector to construct five recombinant plasmids(Mu0、Mu1、Mu2、Mu3 and Mu4).These five recombinant plasmids were transfected into the pancreatic cancerBxPC-3 cell lines and their expressions of green fluorescent protein were observed by fluorescence microscope.Result:(1) The restriction enzyme analysis and nucleotide sequence determination proved that five recombinant plasmids were successfully constructed.(2) The green fluorescent proteins could be observed by fluorescence microscope and hIL-18 expression be detected by RT-PCR in BxPC-3 transfected with five different mutants.Conclusion:(1) Five recombinant plasmids expressing hIL-18 isomers have been successfully constructed.(2) The hIL-18-GFP fusion proteins could be expressed in transfected BxPC-3 cells with five recombinant plasmids pEGFP-C1-hIL-18. |
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| Keywords: | pEGFP-C1 hIL-18 BxPC-3 cells mutants |
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