论文部分内容阅读
目的构建广州管圆线虫Ⅴ期幼虫cDNA文库,免疫学筛选抗原基因。方法提取广州管圆线虫Ⅴ期幼虫总RNA,用Clontech公司的SMARTTMcDNA文库构建试剂盒构建未扩增文库,检测未扩增文库滴度和重组率后,进行文库扩增。用大鼠感染血清作免疫探针筛选文库,PCR扩增外源基因插入片段并测序,用生物信息学软件进行同源性比对,推导氨基酸序列,预测其理化性质。结果成功构建了广州管圆线虫Ⅴ期幼虫cDNA文库,免疫学筛选获得11个阳性克隆,对测序的9个阳性克隆进行初步分析,1个与广州管圆线虫Ⅴ期幼虫的1个EST同源性为99%,6个(有2个为同一克隆)与秀丽隐杆线虫和新杆状线虫均有不同程度的同源性,最高达91%。共有7个阳性克隆存在完整的开放阅读框。结论从广州管圆线虫Ⅴ期幼虫cDNA文库中初步筛选出7个有诊断意义的抗原基因,为其相关研究奠定了基础。
Objective To construct the Ⅴ phase larvae cDNA library of Anemarrhena angularis and screen immunologically the antigenic genes. Methods Total RNA was extracted from stage Ⅴ of C. elegans. The non-amplified library was constructed by using SMARTTM cDNA library from Clontech. The amplification of the library was performed after detecting the titer and recombination rate of the non-amplified library. The rat infected sera was used as an immunological probe to screen the library. PCR was used to amplify the exogenous gene fragment and sequenced. Bioinformatics software was used for homology comparison and deduced amino acid sequence to predict the physicochemical properties. Results The cDNA library of Ⅴ phase larvae of A. cantonensis was successfully constructed. Eleven positive clones were obtained by immunological screening. Nine positive clones were sequenced and one was homologous with one EST of stage Ⅴ of C. elegans Ninety-nine percent (99%) of the sexes, six (two of which had the same clone), had different degrees of homology with Caenorhabditis elegans and new rod-shaped nematodes, up to 91%. A total of 7 positive clones exist in the complete open reading frame. Conclusion Seven diagnostic antigen genes were initially screened from the cDNA library of stage Ⅴ of C. elegans, which laid the foundation for its related research.