猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。

Construction of a Subtractive cDNA Library for Infective Larvae of Ascaris suum

The objective of present study was to construct a subtractive cDNA library for infective larvae of Ascaris suum,for the identification and cloning of differentially expressed genes in infective larvae of A.suum.The subtractive cDNA library for infective larvae of A.suum was constructed with the technique of suppression subtractive hybridization(SSH) using the PCR-Select cDNA Subtraction kit(Clontech) after subtraction with cDNAs representing other developmental stages(adults,eggs,L3 and L4).The specificity of the constructed archives was determined using Southern dot blot,followed by sequencing of 45 random clones showing significant expression in the infective stage.The results showed that the subtractive cDNA library for infective larvae of A.suum was specific highly as confirmed by Southern dot blot.40 of the 41 valid expression sequence targets(ESTs) had significant similarities with that of other known genes,mainly representing L3 genes and female head gene of A.suum.One EST may represent a new gene.The successful construction of the subtractive cDNA library for infective larvae of A.suum provides a foundation for further studies of genes related to the infectivity and migration of A.suum larvae.