猪蛔虫性别特异基因在第三、四期幼虫的表达谱研究

为研究猪蛔虫性别特异基因在第三期、第四期幼虫的表达情况，本研究采用表达谱基因芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库中分别挑取克隆，制备成cDNA微阵列芯片、标记有荧光素Cy5-dUTP和Cy3-dUTP的各组探针（L3＋♀；L3＋♂；L4＋♀；L4＋♂）分别与制备好的芯片进行杂交、扫描，原始信号值经均一化处理后，获取每个点的Ratio值（Ratio=Cy5／Cy3）．根据该值筛选出表达差异最明显的841个克隆，进行测序分析，在获得的707个有效序列中有61个是猪蛔虫新的ESTs、将在第三、四期幼虫以及成虫中显著表达的克隆进行排列组合比较，获得各期特有和各期之间共有的表达克隆一在第三期幼虫中，雌、雄性别特异基因分别有21和9个，编码的主要蛋白有卵黄原前导蛋白、睾丸幼胚蛋白等；在第四期幼虫中特异表达的雌、雄性别基因分别有22和6个，其中免疫抑制卵巢信息蛋白、主要精子纤维蛋白（MFP）等表达明显本项研究结果不仅初步阐明了猪蛔虫性别特异基因在第三、四期幼虫的表达情况，而且亦为筛选控制猪蛔虫病的“关键”基因并阐明其功能奠定了基础。

Expression profiles of gender-specific genes in third and fourth stage larvae of Ascaris suum

Individual positive clones were selected from the male and female A.suum cDNA libraries constructed using of the suppression subtractive hybridization(SSH),each clone was verified and robotically printed onto aminosilane-coated glass slides,and each clone was repeated once in the same slide.Fluorescent cDNA probes were prepared from larvae third(L3),larvae forth(L4)and adult of Ascaris suum.Hybridization experiments were carried out according to the different mix of probes:L3 and adult female,L3 and adult male,L4 and adult female,and L4 and adult male.The hybridisation signal of each spot was measured,normalized,and differential expression was determined according to their Ratios(Ratio=Cy5/Cy3,less 0.5 or more 2),these significantly differentially expressed clones 882 were sequenced.A total of 707 valid ESTs were obtained and 61 of which were considered to represent new genes.In order to obtain stage-specific gender genes,compared clones which distinctly expressed in different development stages(the third and fourth stage and adult).21 female and 9 male clones showed differential expression and in the third stage larvae,and 22 female and 6 male clones showed highly expression in the fourth stage larvae.ESTs representing genes encoding third-specific vitelline precursor and testis germinal zone,and fourth-specific Immunosuppressive ovarian message protein and major sperm fibrin protein were abundant and showed significant expressed.The result revealed expression profiles of gender-specific genes in the L3 and L4 stage of Ascaris suum,furthermore,the experiment identified key gender functional genes and provides foundation for further control Ascariasis.