皱纹盘鲍肝和肾cDNA文库的构建及免疫相关基因的初步分析

Construction of cDNA library of liver and kidney from abolone （Haliotis discus Hannai ） and preliminary analysis of immune gene

用鳗弧菌（Vibrio anguillarum）人工感染皱纹盘鲍（Haliotis discus Hannai）,取等量肝和肾组织混匀,Unizol提取总RNA。用Oligotex mRNA Kits分离mRNA和带有Oligo dT（18）的引物合成双链cDNA,将双链cDNA与PBluescript Ⅱ SK（＋）载体连接后电转化E.coli DH10B,得到cDNA文库。经测定库容为5.24×105cfu/mL,插入片段大小为0.8～2.5kb,构建的文库质量较高。对文库进行测序和生物信息学初步处理,得到了高质量的EST（expressed sequence tags）1282条。用BlastX和BlastN进行序列同源性对比,发现有393个（45%）基因聚类在蛋白质和核酸水平上找到了同源序列。对具有同源序列的基因进行了分析,发现了31种与免疫相关的基因,如防御素、凝集素、基质金属蛋白酶、铁蛋白和热激蛋白等。这些基因均已在Genbank中注册。

The cDNA library of the liver and kidney from the Vibrio anguillarum-infected Haliotis discus Hannai was constructed. Total RNA was extracted using Unizol and mRNA was separated by Oligotex mRNA Kits. Double-strand cDNA was synthesized and inverted into PBluescript ⅡSK（ ＋ ）. The ligated cDNAs were transformed into DHIOB. The content of the library was 5.24×105cfu/mL and the sizes of the inserted fragments ranged from 0.8 kb to 2.5 kb. After random selection of the clones for sequencing, 1282 good qualitive ESTs were obtained. By using software of BlastX and BlastN, 393 （45.02%） ESTs shared significant homology with known sequences in protein or nucleotide database of NCBI. Many genes related to immunology such as defensin, lectin, metallopmteinase, ferritin, Hsp and so on were obtained.