猪IgG Fc受体与恒定链的细胞定位及相互关系研究  

Intracellular localization and association of porcine Fc receptor for IgG with invariant chain

作　　者：吴胜国 倪子豪 李吕木[1] 丁小玲[1] 许发芝[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《中国兽医学报》2016年第3期401-406,453,共7页Chinese Journal of Veterinary Science

基　　金：安徽省高校自然科学研究重点资助项目(KJ2015A078);国家自然科学基金资助项目(30671537)

摘　　要：克隆猪IgG Fc受体基因(FcRn)及其剪接体基因,研究其与猪恒定链(Ii)的细胞定位及相互关系。利用Triozl法从猪十二指肠组织提取总RNA,进行RT-PCR扩增,PCR产物与pMD-18T载体连接,筛选阳性克隆并序列测定和分析。进一步构建了融合表达绿色荧光蛋白的FcRn基因及其剪接体真核表达载体,利用脂质体Lipofectamine2000介导法与能表达红色荧光的Ii真核表达载体共转染COS-7细胞,共聚焦荧光显微镜检测FcRn基因与猪Ii链在细胞中的共定位,并通过免疫共沉淀进一步研究它们之间的相互关系。序列分析结果表明,FcRn基因序列大小为1 071bp,剪接体片段大小为795bp,两者氨基酸序列均含有胞外结构域、跨膜区和胞浆尾区。细胞定位研究显示,LFC-GFP或SFC-GFP与RFP-Ii共转染COS-7细胞后共定位在细胞的内膜系统。免疫共沉淀结果显示,GFP-Ii与LFC-GFP或SFC-GFP共转染后,检测到LFC-GFP或SFC-GFP的表达。猪Ii链与FcRn基因或剪接体能够形成复合体,共定位于细胞的内膜系统。To clone Fc receptor for IgG（FcRn）gene and its alternatively spliced variant of pig,and investigate the intracellular localization and association with pig invariant chain.Total RNA was extracted from mucosal tissue of duodenum by Triozl and mRNA sequences of genes were amplified by RT-PCR.The PCR products were ligated into the pMD-18 Tvector.The recombinant plasmids were screened and the genes were sequenced and analyzed.Furthermore,we constructed the eukaryotic expression plasmids of FcRn and its alternatively spliced variant that were fused with green fluorescent protein.The recombinant plasmids containing FcRn or its alternatively spliced variant and pMcherry-C1-Ii that could express red fluorescent protein were transiently transfected into COS-7cells with Lipofectamine 2000.Confocal immunofluorescence microscopy was carried out to detect the expression and intracellular localization of Ii,FcRn and its alternatively spliced variant.Sequence analysis results showed that the size of FcRn sequence is 1 071 bp,and the size of its alternatively spliced variant is 795 bp.Both contain an extracellular domain,a transmembrane region and a cytoplasmic tail region.Cellular localization studies showed that the colocalization was found in endomembrane system when LFC-GFP or SFC-GFP and RFP-Ii were co-expressed in COS-7cells.Immunoprecipitation of Ii showed that GFP-Ii co-isolated with FcRn and its alternatively spliced variant when COS-7cells were transiently transfected with pEGFP-C1-Ii,pEGFPN1-LFC and pEGFP-N1-SFC.Porcine Ii and FcRn gene or its alternatively spliced variant can form the complexes that are colocalized in endomembrane system of COS-7cells.

关 键 词：猪 FCRN 剪接体 序列分析 细胞共定位 

分 类 号：S852.4[农业科学—基础兽医学]