I 雞的CD3基因之選殖及核甘酸序列分析 II 人類T細胞接受器α、δ倆鏈基因之表現性質研究

Abstract

本論文的第一部份是雞的CD3基因的選殖及序列分析。根據雞CD3 cDNA選殖體T11.15的核甘酸序列合成若干引子，以胸腺cDNA為範本進行聚合脢連鎖反應。其產物包括一個屬於T11.15的DNA片段，CD3 339，及一個T11.15的in-frame deletion的形式，CD3 645。次以genomic DNA為範本進行聚合脢連鎖反應，發現有三個DNA片段，大小分別為700bp, 340bp, 220bp，與T11.15及CD3 645基因有關。由南方墨跡分析得知T11.15/CD3 645的基因大小應不超過4kb。篩選liver genomic library後，我們得到一可能涵蓋了整個基因的DNA片段，約4.0kb 。經次選殖及DNA定序，我們分析此基因轉錄子及插入子的構造，並發現YXXL/I containing motif分佈在兩個轉錄子上。 本論文的第二部份是T細胞接受器α,δ鏈基因之表現性質研究。我們以人類的胸腺及脾臟的cDNA為範本，以3個Vα引子，5個Vδ引子，Cα引子，Cδ引子不同的組合方式進行聚合脢連鎖反應。除了Vα15與Cδ這種組合外，其餘組合方式，在胸腺及脾臟中均有200－500bp的DNA片段出現，說明瞭Vα－Cδ，Vδ－Cα的重組方式在mRNA的層次上也可以發生。這些片段經次選殖及DNA定序，用以分析了J的使用及接和區域的歧異度。

Part I: Base on the sequence of chicken CD3 cDNA clone, T11.15, we synthesized several primers. These primers were used to perform PCR amplication which included thymus cDNA as template. The PCR products contained CD3 339, derived from the T11.15, and CD3 645, an in-frame deletion form of T11.15. Analysis of the PCR-amplified genomic DNA, we found three DNA fragmemts (700 bp,340 bp,220 bp) relative to the T11.15/ CD3 645 gene. The result of Southern bloting implied that the size of T11.15 /CD3 645 gene was no more than 4.0 kb. After screening of genomic library, we obtained a BamHI 4.0 kb fragment which might contain the T11.15 / CD3 645 gene. By subcloning and DNA sequencing, we analysed the exon-intron organization and found the YXXL/I containing motif was encoded by two exons. Part II By combining of three Vα primers, five Vδ primers, Cα primer and Cδprimer in different way, we performed the PCR amplication, using human spleen and thymus cDNA as template. Except the combination of Vα 15 and Cδ, we obtained major bands between 200bp and 500bp. It means the combination of Vα-Cδ or Vδ-Cα is possible in mRNA level. After sub cloning and sequencing of these fragments, we analysed the J chain usage and the junction region diversity.