鲤免疫应答相关基因的克隆与鉴定  

Cloning and identification of immune response related genes in com-mon carp,Cyprinus carpio L.

作　　者：丰培金[1] 王文东[2] 李伟[2] 卢强[2] 

机构地区：[1]临沂大学生命科学学院,山东临沂276005 [2]吉林大学人兽共患病研究所,吉林长春130062

出　　处：《中国水产科学》2011年第5期1179-1188,共10页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(30972277)

摘　　要：为研究鲤(Cyprinus carpio L.)白细胞免疫应答相关的分子机理,以体外培养的鲤外周血白细胞为实验材料,用荧光标记的mRNA差异显示(FluoroDDRT-PCR)技术,研究丝裂原(50μg/mL LPS、50μg/mL PHA和50μg/mL ConA)在刺激白细胞4、12和24 h内诱导白细胞免疫应答相关基因的mRNA表达差异,共获得92个差异片段,其中87个片段有再扩增产物,再扩增率为94.6%;将差异片段克隆,经PCR鉴定,获得81个阳性克隆,鉴定率为93.1%;差异片段序列同源性功能分析结果表明,本研究共获得3个免疫应答相关的cDNA克隆,它们分别编码鲤的蛋白酶体激活因子PA28α亚基、翻译延伸因子(EF-1α)和基质金属蛋白酶13(Mmp13)部分序列,为进一步研究这些差异表达基因在鱼类免疫中的作用机制奠定了基础。mRNA differential-display reverse-transcription polymerase chain reaction（DDRT-PCR） is an effective and quick method to study gene different expression in the same cell under different physiological status and dif-ferent stages of growth and development.In order to study immune response related genes in carp leucocytes,fluorescence DDRT-PCR was used to compare mRNA from leucocytes from peripheral blood of carp with LPA（50μg/mL）,ConA（50 μg/mL） and PHA（50 μg/mL） stimulation and non mitogens stimulation in different time such as 4h,12h and 24h.The results as following,92 different fragments were obtained altogether,of which the re-amplified fragments were found in 87 different cDNAs and the re-amplification rate was 94.6%.Cloning and PCR testing showed that 81 fragments were positive and the positive rate was 93.1%.Analysis with BLAST and DNATools software revealed 3 cDNA fragments were immune response related genes which encoded proteasome activator complex PA28？？subunit,translation elongation factor-1？（EF-1？） and matrix metalloproteinase 13（Mmp-13） of common carp.Bioinformatics analysis showed that the genes encoded by these different fragments were involved in various functions such as MHC class I antigen,signal transduction,translational control,apop-tosis,degradation of the extracellular matrix.It is essential for further studying the mechanisms of these differen-tially expressed genes in fish.

关 键 词：荧光mRNA差异显示 鲤 白细胞 免疫应答相关基因 

分 类 号：S94[农业科学—水产养殖]