CD8α，點帶石斑魚細胞毒殺T淋巴球表面標記，cDNA選殖與定性

CD8是表現在毒殺性T淋巴球表面的醣蛋白分子，可連接第一型主要組織相容複合體(MHC class I)，幫助毒殺T淋巴球摧毀被病毒感染或腫瘤細胞。從點帶石斑魚的胸腺選殖出CD8α cDNA，包括43 bp的5’端非載碼區、684 bp的載碼區，和不同長度333、534和853 bp的3’端非載碼區，分別命名為CD8α S、CD8α M和CD8α L。載碼區轉譯出含227個胺基酸的蛋白質，含一段訊息胜肽、一段免疫球蛋白家族區塊、一段免疫球蛋白關節區塊、一段穿膜區塊和一段細胞質尾區塊；其中細胞質尾區塊有一段和酪胺酸激酶p56lck的結合位。蛋白質分子量推測為24 kDa、等電點9.62。系統分類與分子演化分析顯示石斑魚和其他海水魚的CD8α一致性在50~60%。Q-PCR 檢測CD8α分佈在胸腺、頭腎、鰓、脾臟和腸，不論 S、M或L型都以胸腺表現最多，且以M型的表現最多。石斑魚注射神經壞死病毒後四天，Q- PCR檢測CD8α在脾臟的表現顯著高於未注射組，屬於可誘發基因。利用昆蟲細胞桿狀病毒表現系統合成CD8α重組蛋白，經SDS-PAGE和西方墨點轉漬法檢驗，分子量約為22.5 kDa。CD8α重組蛋白免疫紐西蘭大白兔九週後取得抗血清，使用免疫細胞化學染色，抗血清可辨識部分週邊白血球，細胞質呈環型戒指狀、高核質比、直徑約7 μm，推測為淋巴球次群。石斑魚注射神經壞死病毒一週後，抽血製備週邊白血球，再利用磁珠正篩選計數CD8α+淋巴球從免疫前3.6~5.5% 增加至免疫後4.2~6.68%。石斑魚注射神經壞死病毒一週後，CD8α+淋巴球會毒殺被神經壞死病毒感染、相同MHC基因型鰭細胞，毒殺活性顯著高於被嘉納魚虹彩病毒感染、相同MHC基因型的鰭細胞或是神經壞死病毒感染、不同MHC基因型的鰭細胞。綜合以上結果顯示被CD8α抗體辨識的淋巴球具專一毒殺活性，受主要組織相容複合體限制，推測屬細胞毒殺性T淋巴球。點帶石斑魚CD8α抗體不會辨識黑鯛、日本鰻和吳郭魚的週邊白血球。此CD8α抗體可作為研究點帶石斑魚細胞性免疫反應的工具。

關鍵字

CD8α cDNA ；細胞毒殺T淋巴球；主要組織相容複合體限制；神經壞死病毒

並列摘要

CD8 is a heterodimer membrane glycoprotein found primarily on the surface of cytotoxic T lymphocytes. It helps the CTLs to kill the infected cells by binding to MHC class I protein that present antigens on the infected-cell surface. In the present study, we cloned the full length cDNAs from orange-spotted grouper (Epinephelus coioides), which included 43 bp 5’UTR, 684 bp ORF and varied length of 333, 534 and 853 bp 3’UTR, and were named as CD8α S, M and L, respectively. The deduced protein of CD8α is 227 amino acid residues long and included one signal peptide, Ig superfamily domain, hinge region, transmembrane domain and cytoplasmic tail and a conserved binding motif associate with tyrosine kinase p56lck . The MW is estimated as 24 kDa with a pI of 9.62. Phylogenetically, the predicted grouper CD8α protein is similar to CD8α from other marine fish species, and the identity is 50-60％. Q-PCR revealed that CD8α is constitutively express in thymus, head kidney, gill, spleen and gut. Optimal expression was found in thymus for both CD8α S, M and L forms, and the CD8α M transcript is the most abundant. The Q-PCR results revealed that the CD8α in the spleen of NNV-injected fish was significantly up-regulated in 4 days post-injection compared to the untreated fish. Moreover, the recombinant CD8α protein was biosynthesized in insect cell-baculovirus expression system examined the MW was 22.5 kDa via the SDS-PAGE and Western blotting . The antiserum prepared from New Zealand white rabbit against biosynthesized recombinant CD8α protein could recognize the subset lymphocytes specifically from PBL with characteristics of 7μm in diameter, high nucleus/cytoplasm ratio and the ring-shaped cytoplasm. After NNV injection for one week, the CD8α+ lymphocytes were proliferated from 3.6~5.5% to 4.2~6.68% which was counted after dynabeads positive selecton. The cytotoxic activity of CD8α+ lymphocytes at one-week post-NNV injection enhanced significantly against NNV-infected syngeneic fin cells in comparison with NNV-infected allogeneic or RSIV-infected syngeneic fin cells. These results revealed that the specific cytotoxicity and MHC restriction of the CD8α antibodies recognized-lymphocytes are the CTLs. The CD8α antibodies can’t recognize the PBLs of black sea bream, japanese eel and tilapia, however it can only specifically recognize the PBLs of the spotted grouper. Conclusively, the CD8α antibodies can provide a tool to advance the research in cellular immune response of spotted grouper.

並列關鍵字

CD8α cDNA ； cytotoxic T lymphocyte ； MHC restriction ； nervous necrosis virus

參考文獻

林清龍、吳慶麗、陳秀男。石斑魚病害與處理。1997。行政院農業委員會。

楊玉婷、陳葦芋、陳政忻。石斑魚產業概況及趨勢。2009。台灣經濟研究院生物科技產業研究中心。

Addison EG, North J, Bakhsh I, Marden C, Haq S, Al-Sarraj S, Malayeri R, Wickremasinghe RG, Davies JK, Lowdell MW. Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity. Immunology. 2005 116:354–361

Araki K, Akatsu K, Suetake H, Kikuchi K, Suzuki Y. Characterization of CD8+ leukocytes in fugu (Takifugu rubripes) with antiserum against fugu CD8α. Developmental & Comparative Immunology. 2008 32:850–8.

Barber EK, Dasgupta JD, Schlossman SF, Trevillyan JM, Rudd CE. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl Acad Sci USA. 1989 86:3277–81.

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CD8α，點帶石斑魚細胞毒殺T淋巴球表面標記，cDNA選殖與定性

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