豬IgG Fc受體與恒定鏈的細(xì)胞定位及相互關(guān)系研究
發(fā)布時間：2018-09-09 15:27
【摘要】：克隆豬IgG Fc受體基因(FcRn)及其剪接體基因,研究其與豬恒定鏈(Ii)的細(xì)胞定位及相互關(guān)系。利用Triozl法從豬十二指腸組織提取總RNA,進(jìn)行RT-PCR擴(kuò)增,PCR產(chǎn)物與pMD-18T載體連接,篩選陽性克隆并序列測定和分析。進(jìn)一步構(gòu)建了融合表達(dá)綠色熒光蛋白的FcRn基因及其剪接體真核表達(dá)載體,利用脂質(zhì)體Lipofectamine2000介導(dǎo)法與能表達(dá)紅色熒光的Ii真核表達(dá)載體共轉(zhuǎn)染COS-7細(xì)胞,共聚焦熒光顯微鏡檢測FcRn基因與豬Ii鏈在細(xì)胞中的共定位,并通過免疫共沉淀進(jìn)一步研究它們之間的相互關(guān)系。序列分析結(jié)果表明,FcRn基因序列大小為1 071bp,剪接體片段大小為795bp,兩者氨基酸序列均含有胞外結(jié)構(gòu)域、跨膜區(qū)和胞漿尾區(qū)。細(xì)胞定位研究顯示,LFC-GFP或SFC-GFP與RFP-Ii共轉(zhuǎn)染COS-7細(xì)胞后共定位在細(xì)胞的內(nèi)膜系統(tǒng)。免疫共沉淀結(jié)果顯示,GFP-Ii與LFC-GFP或SFC-GFP共轉(zhuǎn)染后,檢測到LFC-GFP或SFC-GFP的表達(dá)。豬Ii鏈與FcRn基因或剪接體能夠形成復(fù)合體,共定位于細(xì)胞的內(nèi)膜系統(tǒng)。
[Abstract]:The porcine IgG Fc receptor gene (FcRn) and its splice gene were cloned and their cellular localization and relationship with porcine (Ii) were studied. Total RNA, was extracted from pig duodenum by Triozl method. The product of RT-PCR amplification was ligated with pMD-18T vector. The positive clones were screened and sequenced. Furthermore, the eukaryotic expression vector of FcRn gene and splice was constructed. The eukaryotic expression vector was co-transfected into COS-7 cells by liposome Lipofectamine2000 mediated and Ii eukaryotic expression vector expressing red fluorescence. The co-localization of FcRn gene and porcine Ii chain in cells was detected by confocal fluorescence microscopy, and the relationship between them was further studied by immunoprecipitation. The results of sequence analysis showed that the size of FcRn gene was 1 071bpand the splice fragment was 795bp. both amino acid sequences contained extracellular domain, transmembrane region and cytoplasmic tail region. The study of cell localization showed that LFC-GFP or SFC-GFP co-transfected COS-7 cells with RFP-Ii and then co-located in the intimal system of the cells. The results of co-immunoprecipitation showed that the expression of LFC-GFP or SFC-GFP was detected after co-transfection of GFP-Ii with LFC-GFP or SFC-GFP. Porcine Ii strand and FcRn gene or splice can form a complex, which is located in the endomembrane system of the cell.
【作者單位】： 安徽農(nóng)業(yè)大學(xué)動物科技學(xué)院;
【基金】：安徽省高校自然科學(xué)研究重點(diǎn)資助項目(KJ2015A078) 國家自然科學(xué)基金資助項目(30671537)
【分類號】：S828
本文編號：2232773
[Abstract]:The porcine IgG Fc receptor gene (FcRn) and its splice gene were cloned and their cellular localization and relationship with porcine (Ii) were studied. Total RNA, was extracted from pig duodenum by Triozl method. The product of RT-PCR amplification was ligated with pMD-18T vector. The positive clones were screened and sequenced. Furthermore, the eukaryotic expression vector of FcRn gene and splice was constructed. The eukaryotic expression vector was co-transfected into COS-7 cells by liposome Lipofectamine2000 mediated and Ii eukaryotic expression vector expressing red fluorescence. The co-localization of FcRn gene and porcine Ii chain in cells was detected by confocal fluorescence microscopy, and the relationship between them was further studied by immunoprecipitation. The results of sequence analysis showed that the size of FcRn gene was 1 071bpand the splice fragment was 795bp. both amino acid sequences contained extracellular domain, transmembrane region and cytoplasmic tail region. The study of cell localization showed that LFC-GFP or SFC-GFP co-transfected COS-7 cells with RFP-Ii and then co-located in the intimal system of the cells. The results of co-immunoprecipitation showed that the expression of LFC-GFP or SFC-GFP was detected after co-transfection of GFP-Ii with LFC-GFP or SFC-GFP. Porcine Ii strand and FcRn gene or splice can form a complex, which is located in the endomembrane system of the cell.
【作者單位】： 安徽農(nóng)業(yè)大學(xué)動物科技學(xué)院;
【基金】：安徽省高校自然科學(xué)研究重點(diǎn)資助項目(KJ2015A078) 國家自然科學(xué)基金資助項目(30671537)
【分類號】：S828
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 萬博;豬繁殖與呼吸綜合征病毒感染后免疫抑制與IgG Fc受體轉(zhuǎn)錄[D];河南農(nóng)業(yè)大學(xué);2010年
，本文編號：2232773
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2232773.html
最近更新
教材專著
