文昌鱼AmphiDC-like的全长cDNA克隆及其表达模式分析

Cloning and Expression Pattern Analysis of AmphiDC-like in Amphioxus

本文旨在克隆文昌鱼树突样蛋白基因AmphiDC-like,采用实时PCR法和原位分子杂交法对其表达模式进行分析.从文昌鱼神经胚cDNA文库测序得到的ESTs中筛选得到该基因片段,通过引物步移直接测序的方法,克隆得到其cDNA全长序列,对其推测的氨基酸序列进行同源性分析发现,该基因产物具有树突样细胞蛋白共有的保守区,并与多种生物的树突状细胞蛋白具有高度同源性,其中与脊椎动物的树突样细胞蛋白同源性较高.实时PCR结果显示,AmphiDC-like在文昌鱼受副溶血性弧菌(Vibrio parahe molyticus,Vp)感染后6h到48h表达均上调,并通过原位分子杂交技术观察到,该基因在文昌鱼鳃和消化道中有表达,为深入研究其功能奠定了基础.

AmphiDC-like has a close relationship to human dendritic cell proteins. The full length of AmphiDC-like of Qingdao amphioxus neurula was cloned from a cDNA library by primer walking approach, then sequenced. The deduced amino acid sequence were analyzed and compared with its homologs in Caenorhabditis elegans , Drosophila melanogaster , Danio rerio , Xenopus laevis , Mus musculus , Gallus gallus, Pan troglodytes and Homo sapiens. The results have shown that the obtained AmphiDC-like contained an open reading frame encoding a putative protein of 375 amino acid residues with a PCI domain that was highly conserved in the dendritic cell proteins among other species. The phylogenetic analysis exhibited a highdegree conservation of the AmphiDC-like in vertebrates. The gene AmphiDC-like was found to be expressed in the intestine and the gills by in situ hybrization and could be up-regulated 6 to 48 hours after the Vibrio Parahemolyticus （ Vp ） challenge as determined by real-time PCR analysis. Our findings will facilitate the subsequent investigations of the amphioxus AmphiDC-like fuctions.