摘要
目的构建田鼠巴贝虫cDNA文库，从中筛选免疫诊断候选抗原。方法用田鼠巴贝虫感染BALB／c小鼠．约7d后待虫血密度达到70％时取血．提取虫体总RNA．经mRNA纯化试剂盒纯化后，构建田鼠巴贝虫的cDNA文库。用田鼠巴贝虫感染阳性小鼠血清筛选cDNA文库．获得阳性克隆，PCR鉴定阳性克隆插入片段大小．测序并进行同源性分析。结果所构建的田鼠巴贝虫cDNA文库的滴度为5．2×10^5噬菌斑形成单位（plaqueforming unit，pfu）／ml，重组率为91．0％，文库插入片段平均长度为1．1kb。从cDNA文库中筛选获得Bm2、Bm4、Bm6、Bm7、Bm9和Bml5等6个阳性克隆，插入片段依次约为633、614、1073、890、1001和1032bp，6个片段均包含开放阅读框，依次编码159、100、254、217、60和244个氨基酸，相对分子质量（尬）依次约为17900、11400、28600、24000、6800和28900。结论构建了田鼠巴贝虫eDNA文库．并筛选获得6个能被感染阳性小鼠血清识别的田鼠巴贝虫阳性克隆，为进一步筛选田鼠巴贝虫免疫诊断抗原奠定了基础。
Objective To construct a cDNA library of Babesia microti and immunoscreen candidate antigens for immuno-diagnosis of Babesia infection. Methods The blood samples were collected from the mice infected with B. microti 7 days post infection when the parasitemia density reached 70%. Total RNA of B. microti was extracted and the mRNA obtained by a purifying kit was used to construct the cDNA-library. The library was immunoscreened with pooled sera from B. microti infected mice to obtain positive clones. The inserted fragments of positive clones were identified by PCR amplification, and the obtained genes were sequenced and analyzed for their homology. Results The titer of the library was 5.2 ×10^5 plaque forming unit （pfu）/ml. The inserted fragment length of the library ranged from 500 to 3 000 bp with a recombination efficiency being 91.0%. Six positive clones, Bm2, Bm4, Bm6, Bm7, Bm9 and Bml5 were found and their inserted fragment length was about 633, 614, 1 073, 890, 1 001 and 1 032 bp, respectively. Sequence analysis revealed that all the 6 clones contained open reading frames. The deduced amino acid sequences of the 6 clones contained 159, 100, 254, 217, 60 and 244 amino acid residues, with Mr of 17 900, 11 400, 28 600, 24 000, 6 800 and 28 900, respectively. Conclusion A cDNA library of B. microti has been constructed and 6 positive clones identified, which can be recognized by the sera of mice infected with B. microti. This study provided preliminary information for further identification of highly reactive antigens for development of immunodiagnostics of B. microti infection.
出处
《国际医学寄生虫病杂志》
CAS
2013年第3期130-134,共5页
International JOurnal of Medical Parasitic Diseases
基金
卫生行业科研专项经费资助项目（201202019）
