犬弓首蛔虫雄虫cDNA文库构建及EST分析

为构建T.canis雄虫cDNA文库,采用Trizol法提取T.canis雄虫的总RNA,合成cDNA,连接到λTripEx2载体上,通过包装蛋白对连接产物的包装,接种到大肠杆菌XL-1-Blue中进行原始文库和扩增文库的滴度测定.经质量鉴定表明:初始文库的滴度为5.25×106 pfu·mL-1,扩增后文库的滴度为6.90×109 pfu ·mL-1.文库的插入片段大小在500～2 000 bp,平均片段大小为1 000 bp,重组率为99.47％.所有指标均显示已成功构建了T.canis雄虫的cDNA文库.利用该文库获得了189条5'有效表达序列标签(EST).对ESTs拼接后代表了101个Unigenes,含有27个Contigs和74个Singletons.其Unigenes在GenBank中的序列号为HO348195～HO348295.同源性分析检索到有56个Unigenes与已知基因同源,其中具有已知或推测功能的基因有40个,未知功能基因有16个,未比对上的基因45个.未比对上的基因与NR数据库中的蛋白序列没有任何意义的匹配,为研究中发现的新基因.这些结果为进一步开展犬弓首蛔虫功能基因及分子机制研究奠定了基础.

Construction of a cDNA Library from Male Adult Toxocara canis and Analysis of Expressed Sequence Tag (EST)

To construction of a cDNA library for male adult T.canis,total RNA were extracted using Trizol reagent from Invitrogen(GIBCO BRL) according to the manufacturer’s instruction.The cDNA was synthesized and the ds cDNA was ligated into the λTripEx2 Vector.The ligation products were packaged with Gigapack III Gold Packaging Extract(Stratagene).Then the mixtures were transformed into Escherichia coli XL-1-Blue host cell culture to determine the titers of the unamplified and amplified libraries.The library qualification evalution showed: the titer of the primary cDNA library was 5.25×106 pfu·mL-1 with a recombination rate of 99.47%,and the titer of the amplified cDNA library was 6.90×109 pfu·mL-1.PCR amplification of randomly picked clones revealed that the inserted cDNA fragments ranged from 500 to 2 000 bp with an average length of 1 000 bp.The cDNA library was constructed successfully,and 189 efficient ESTs(expressed sequence tags) of 5′ end were obtained from this cDNA library.Cluster analysis of these ESTs identified 101 unique sequences containing 27 Contigs and 74 Singletons.All the unique sequences were deposited under dbEST in GenBank(GenBank: HO348195-HO348295).BLASTX searches revealed that 45 Unigenes(44.55% of the total) or 88 ESTs(46.56% of the total) were novel genes that had no significant matches to any protein sequences in the public databases.The rest 56 Unigenes(55.44% of the total) or 101 ESTs(53.44% of the total) were closely matched to the known genes or sequences deposited in the public databases.This work will provide a valuable resource for further research on gene function and molecule mechanism of T.canis.