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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 for the predominant diagnostic antigen that is highly immunoreactive with the serum of C. elegans infected mice. Methods Serum was used as an immunological probe to screen the cDNA expression library of C. elegans of larvae of Acinetobacter nocturnal. The positive clones were sequenced and bioinformatics analyzed according to the sequencing results Primers were designed to amplify this gene and clone into PET-30a (+) vector. Results A total of 6 positive clones were obtained. DNA sequencing and homology analysis showed that 2 of them were highly homologous to MYT1, 1 of them were closely related to the mRNA of the collagen family A (alpha-collagen) highly homologous, and are full-length cDNA. The full length cDNA of MYT1 was cloned. The constructed recombinant plasmid of the new gene was double digested and proved to contain the target fragment in accordance with the length of the target fragment. CONCLUSION: Two T. rhamnosus genes were screened from infected mouse serum and the prokaryotic expression recombinant plasmid PET30a (+) - MYT1 of MYT1 was successfully constructed.