石斑魚免疫球蛋白M基因之選殖及分子特性

Abstract

石斑魚為亞、熱帶珊瑚礁魚類，是台灣及東南亞地區重要的養殖魚種，肉質鮮美，產量逐年增加，供不應求。由於高密度之養殖方式，造成疾病叢生，為了了解石斑魚的免疫系統以作為防治之基礎，我們進行石斑魚免疫球蛋白M（IgM）的研究。石斑魚IgM是由重鏈與輕鏈組成的四聚體，之前本實驗室已自點帶石斑（Epinephelus coioides）選殖出四型的IgM重鏈基因（Cheng et al., 2006），本研究進一步選殖並分析其輕鏈基因。利用本實驗室選殖出的一段978 bp之IgM輕鏈基因為探針，自石斑魚頭腎cDNA基因庫中選殖出20個全長的石斑魚輕鏈基因。此輕鏈cDNA基因大小介於708至747 bp之間，在5’及3’端具有非轉譯區，其開放讀架區可轉譯為分子量24.6-26.5 kDa之輕鏈。N端leader之後為變異區及保守區。變異區由FR與CDR組成。保守區中含有3個保守性高的cysteine，這些cysteine被認為是輕鏈內部以及其與重鏈之間形成雙硫鍵的橋樑。依據保守區的胺基酸序列，可將這些基因歸納為四種類型，而其變異區則可區分為五種家族。J區連接變異區及保守區，依照J區之N端及C端的序列差異可以推論VJC組合方式具有多樣性，本實驗共找到十種不同的C端序列組合。變異區和保守區與其他已發表的魚類IgM輕鏈基因相比有很高的相似性，依據保守區演化樹分析，我們發現石斑魚具有四種類型IgM輕鏈，比其他魚種的IgM輕鏈類型多。RT-PCR之分析結果，顯示四類型IgM輕鏈在石斑魚頭腎組織中皆有表現。

Groupers, coral fish species that inhabit in tropical and subtropical regions, are critical to Taiwanese aquaculture as well as to the rest of Southeast Asia. Due to the culinary value and the ever-growing demand, the yields of groupers progressively increase year after year. However, the highly populated breeding methods within confined or limited habitation space are prone to induction and prevalence of diseases. In order to strengthen the groundwork of disease control and preventative research via a better understanding of grouper's immune system, we initiated this study concerning immunoglobulin M (IgM) of groupers. Immunoglobulin M is a tetramer that consists of two heavy chains and two light chains. Our previous findings identified and cloned 4 CH families from Epinephelus coioides (Cheng et al., 2006). Based on that result, we further analyzed the light chain genes in this proposed study. Utilizing a 1006 bp light chain gene that developed by our laboratory as a probe, we cloned 20 full-length light chains from grouper's head kidney cDNA library. The length of this light chain gene is between 708 to 747 bp, with a non-translated region at 5' and 3' end. In addition, its ORF can be translated into a light chain with molecular weight of 24.6-26.5 kDa. The variable and constant regions are located behind the leader. The variable region is composed of FRs and CDRs; while the constant region consists of 3 highly constant cysteines. It has been suggested that these cysteines formed intradomain and interchain disulfide bonds. According to the amino acid sequences of the constant region, these genes can be classified into 4 isotypes; while the variable region can be distinguished into 5 families. Based on the difference in N- and C-terminal in J segment, J segment can bridge variable and constant region with varieties of VJC combinations, which make it diversity. Overall, we've found 10 different sequences of C-terminal. According to the phylogenetic analysis of grouper's IgM light chains constant region to that of other fish species, we've found that groupers possess 4 isotypes of IgM light chains, relatively higher than other teleost fish. Furthermore, the result of RT-PCR analysis indicated that there are 4 isotypes of IgM light chains expressed in grouper's head kidney tissue.