广州管圆线虫Ⅴ期幼虫cDNA文库的构建及免疫学筛选

Construction and immunoscreening of specific antigen genes from a Ⅴ stage larvae cDNA library of Angiostrongylus cantonensis

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 and screen a cDNA library of V stage larvae of Angiostrongylus cantonensis to obtain specific antigen genes for diagnosis. Methods Total RNA was extracted from V stage larvae of A. cantonensis. After that, an unamplified library was constructed the using SMARTTM cDNA Library Construction Kit from Clontech Laboratories. The titer and the recombination rate of the unamplified library were detected and then amplified. Positive clones were obtained by using antisera of rats infected with A. cantonensis as an immuno-probe. Some of the inserts of positive clones were specifically amplified by PCR and sequenced. The nucleotides and the prediction of open reading frames （ORFs） and physico-chemical properties of deduced amino acids were analyzed using bioinformatics. Results This study constructed a cDNA library of V stage larvae of A. cantonensis using SMART LD-PCR, and eleven positive clones were obtained through preliminary immunoscreening. The inserts of nine positive clones were sequenced and analyzed for their homology in the GenBank database. Results showed that one EST of V stage larvae of A. cantonensis had homology of 99% and that one was not homologous to genes in GenBank. The maximum homology of the other six sequences, two of which were the same, was 91% in comparison to nucleotides from Caenorhabditis elegans and Caenorhabditis briggsae. There were total seven positive clones with complete ORFs. Conclusion Constructing and immunoscreening a cDNA library of V stage larvae of A. cantonensis yielded seven significant clones, providing a basis for future research on these genes.