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鸡恒定链P31基因及其点突变体在真核细胞中表达与定位特性

发表时间:2009-06-15  浏览量:1566  下载量:716
全部作者: 李林,仲大莲,余为一
作者单位: 安徽农业大学,安徽省人兽共患病重点实验室
摘 要: 为研究鸡恒定链(chicken invariant chain,ChIi)分子免疫调节功能及其机理,用2对引物通过聚合酶链反应(polymerase chain reaction, PCR)从PMD18-T/Ii P31质粒中扩增出ChIi的P31基因,将该基因分别插入真核表达载体pEGFP-N1和pEGFP-C1中,构建成N1-P31和C1-P31质粒。另设计4条突变用引物,利用大引物突变的方法,经2次点突变,构建成C1-P8-18A质粒。经双酶切鉴定和测序鉴定后,将这3个质粒分别转染293细胞株,经48 h培养后用荧光显微镜检测。结果表明:重组质粒N1-P31和C1-P31转染的细胞荧光主要出现在胞浆区的细胞器内,而由C1-P8-18A,pEGFP对照转染的细胞中绿色荧光则主要分布在胞核,这说明融合蛋白ChIi-GFP或GFP-ChIi中ChIi分子在真核细胞中具有胞内转运的生物学功能,且ChIi分子具有内体定位的作用。
关 键 词: 兽医免疫学;免疫基因;鸡恒定链;绿色荧光蛋白;真核表达
Title: Expression and location characteristics of chicken invariant chain P31 gene and its point mutant in eukaryotic cells
Author: LI Lin, ZHONG Dalian, YU Weiyi
Organization: Key Laboratory of Zoonoses of Anhui Province, Anhui Agricultural University
Abstract: To research the molecular immunoregulation function of chicken invariant chain (ChIi) and its mechanism, ChIi P31 gene was amplified from PMD18-T/Ii P31 via PCR using two pairs of primers, and the P31 gene was inserted into pEGFP-N1 and pEGFP-C1 respectively to form two recombinant plasmids, N1-P31 and C1-P31. 4 mutation primers were designed, and two point mutations were applied using large primer mutation method to construct C1-P8-18A plasmids. After double enzyme digestion and sequencing identification, the three plasmids were respectively transfected into human 293 cell strain, and the cells were cultivated for 48 h and then observed by a fluorescent microscope. The results showed that fluorescence of the cells transfected by N1-P31 and C1-P31 mainly appeared in organelles in the cytoplasmic domain, while the green fluorescence of the cells transaffected by the C1-P8-18A and pEGFP mainly distributed in the nucleuses. All these indicate that the ChIi molecules in fusion proteins ChIi-GFP and GFP-ChIi have biological function of transportation in eukaryotic cells, and the ChIi molecules have endosome location function.
Key words: veterinary immunology; immune gene; chicken invariant chain; green fluorescent protein; eukaryotic expression
发表期数: 2009年6月第11期
引用格式: 李林,仲大莲,余为一. 鸡恒定链P31基因及其点突变体在真核细胞中表达与定位特性[J]. 中国科技论文在线精品论文,2009,2(11):1203-1207.
 
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