雞CD8α基因的分離與鑑定

Abstract

CD8分子是一種醣蛋白，由T細胞所表現至細胞膜上。CD8分子主要辨認MHC Class I分子，在CTL(Cytotoxic T Cell)細胞的活化和執行刺殺任務時，扮演加強和目標物結合的親合力，和訊息傳遞的角色。在結構上主要是由α鏈和β鏈以Heterodimer的型式出現，兩條鏈間以雙硫鍵鍵結(l)(30)(31)。 在本篇論文，主要確定雞的CD8α基因，以及CD8α在Genome上的圖譜(Map)，並且加以定序，結果發現CD8α基因分佈在6kb的範圍，六個Exon組成，Exon1是5'Untranslated region和Leader Sequence;Exon2是Ig V-like Region; Exon3是Hinge-like Region；Exon4是Transmembrane Domain；Exon5,6是Cytoplasmic Domain。我們並以雞的CD8α基因和人跟老鼠作比較，發現三者在結構上都很相似，尤其Transmembrane Domain和Cytoplasmic Domain相似性高達70％。從演化的觀點來看，CD8分子是Immunoglobulin Superfamily的一員。CD8分子的V-like Domain和Ig和TCR的V Region部份都有相似性，因此我們以CD8α的V-like Region作探針（probe)，在Genome Blot 上發現很多訊號(Band)，其中很多訊號（Band)，並非CD8α所有，其中有兩段基因片段，大小分別為0.9kb和1.2kb ，位於CD8α上游，我們將它定序，並以此二段基因序列，進入GCG搜尋，發現和人跟老鼠Ig Heavy Chain和Light Chain的V Region很相似，由於在人跟老鼠，Ig k Light Chain和CD8基因位於同一條染色體，而在雞至今仍然未發現Ig k light chain的存在，所以這兩段很類似Ig Vk基因的DNA片段，如果雞有Ig k light chain，則此二段片段很可能是雞的Ig k light chain的一部分；或也有可能是Ig k light chain的祖先，人類和老鼠為了增加Ig的變異性，Ig k light chain才被分化出來，而雞保存了這一個演化上的證據。 我們並以聚合?連鎖反應（PCR）的方式，釣出一段雞的CD8β基因片段，若在雞內α和β的基因結構也像人跟老鼠一樣，是位於同一條染色體，此片段距離CD8α基因至少17kb以上。

The CD8 glycoprotein is expressed on the surface of T lymphocytes .CD8 is thought to bind MHC class I molecules and to function both as an adhesion molecule, stabilizing the interaction of T lymphocyte with their targets, and as a signal transduction unit. CD8 exists as a disulfide-linked heterodimer of the α and β chains. Here we report the complete coding sequence of chicken CD8α gene, and its genomic organization. This gene spans approximately 6kb and is organized into six exons which encode separate functional domains of the protein. ExonⅠencodes the 5'untranslated region and leader peptide, exon Ⅱ the Ig V-like region, exon Ⅲ the hinge region, exon Ⅳ the transmembrane domain, and exon Ⅴ and Ⅵ the cytoplasmic tail. Comparison of the CD8α gene of human, mouse, and chicken has revealed a high homology in structure, especially the transmembrane domain and the cytoplasmic domain (70% homology). CD8 molecules are a member of immunoglobulin gene superfamily. The V-like region of CD8α has homology to Ig V and TCR V region. Using V-like DNA segments of CD8α as probes, we found there are many homologous DNA fragment shown by genomic blot hybridization. After sequencing two of them, named BamHI 1.2k and BamHI 0.9k, which locate upstream of CD8α, we found that they are highly homologous to Ig V-like region of the CD8α gene and to Ig VK region. The amino acid required for the formation of the immunoglobulin fold, such as the two cysteines involved in intrachain disulfide bond formation and the invariant tryptophan, are located in the expected position. There is no report about Ig K light chain of chicken so far. But it is known Ig K light chain are located on the same chromosome in both human and mouse. It is possible BamHI 1.2k and BamHI 0.9k are the ancestor of Ig VK light chain. We amplify a probe of chicken CD8β gene by PCR. The results of southern blot analysis indicated that the distance between the probe and the CD8α gene is greater than 17kb; that is if the CD8α and CD8β are located in the same chromosome as their human and mouse homologs.