广州管圆线虫V期幼虫cDNA表达文库中优势诊断抗原的筛选、鉴定和克隆

Immunoscreening,cloning and primary identification of the potential diagnosis antigen genes from the cDNA library of Angiostrongylus cantonensis,stage-V larvae

目的筛选与广州管圆线虫感染小鼠血清具有较强免疫反应性的优势诊断抗原。方法用感染广州管圆线虫21d的小鼠感染血清做免疫探针筛选广州管圆线虫V期幼虫cDNA表达文库,对筛选得到的阳性克隆进行插入片段的测序和生物信息学分析,再根据测序结果设计引物扩增该基因并将其克隆入PET-30a(+)载体中。结果共获得6个阳性克隆,经DNA测序及同源性分析表明,发现其中2个与广州管圆线虫髓鞘转录因子1(MYT1)高度同源,1个与广州管圆线虫胶原蛋白家族基因(alpha-collagen)高度同源,且均为全长cDNA。克隆出广州管圆线虫MYT1全长cDNA序列,构建的新基因重组质粒经双酶切后证明含有与目的片段长度相符合的目的片段。结论用感染小鼠血清作为探针初步筛选到2个广州管圆线虫基因,并成功构建MYT1的原核表达重组质粒PET30a(+)-MYT1。

Objective To screen and identify potential diagnosis antigen genes from the cDNA library of A ngiostrongylus cantonensis stage-V larvae. Methods A.cantonensis stage-V larvae cDNA library was screened with anti-sera of mice 21 days post A.cantonensis infection. The inserts of the positive clones were sequenced and analysed by bioinformatics. According to sequencing results, primers were designed to amplify the gene and cloned into vector PET-30a（＋）. Results Six positive clones were obtained through repeated immunoscreening. Two of which were highly homologous to the putative myelin transcription factor 1 （MYT1）,and one was highly homologous to the putative alpha-collagen. A full-length cDNA sequence of MYT1 was cloned and identified. Conclusions Two novel genes of A .cantonensis were identifed. The rfecombinant prokaryotic expression plasmid PET30a（＋）-MYT1 was successfully constructed.