肝片吸虫诊断候选抗原的筛选

旨在筛选出肝片吸虫诊断候选抗原,拓展诊断肝片吸虫感染引起的肝吸虫病的靶点。通过蛋白免疫印迹试验从肝片吸虫成虫噬菌体展示cDNA文库中筛选出肝片吸虫特异性抗原基因,对阳性克隆进行测序和生物信息学分析。克隆特异性抗原基因,并连接表达载体pET-32a(+),构建重组质粒,转化至BL21(DE3)感受态细胞,通过IPTG诱导重组蛋白表达,用SDS-PAGE和Western-blot验证其表达情况。结果表明,共计获得了18个阳性克隆。通过候选抗原核酸序列同源性比较,发现肝片吸虫假定蛋白D915000758具有完整的开放阅读框。经生物信息学分析,发现该蛋白cDNA全长为345 bp,编码114个氨基酸。成功扩增出该特异性抗原基因,SDS-PAGE结果表明重组蛋白pET-Fh以包涵体形式表达,分子质量大小约为32 ku;Western-blot证明该重组蛋白可以被肝片吸虫阳性血清特异性识别。结果提示,假定蛋白D915000758可作为潜在的肝片吸虫病诊断候选抗原,应用于肝片吸虫感染引起的肝吸虫病的诊断技术中。

Screening of candidate antigens for diagnosis of Fasciola hepatica

The purpose of this study was to screen out candidate antigens for the diagnosis of Fasciola hepatica and expand the target for the diagnosis of liver fluke disease caused by F.hepatica infection.The F.hepatica specific antigen gene was selected from the F.hepatica adult phage display c DNA library by immunoblot assay,and the positive clones were sequenced and analyzed by bioinformatics.The specific antigen gene was ligated into the expression vector pET-32 a(+),and the recombinant plasmid was constructed and transformed into BL21(DE3)competent cell.The expression of recombinant protein was induced by IPTG and verified by SDS-PAGE and Western-blot.The results showed that 18 positive clones were successfully screened.The homology comparison of candidate antigen nucleic acid sequences revealed that hypothetical protein D915000758 has a complete open reading frame.Bioinformatics analysis showed that the full-length c DNA of the protein D915000758 was 345 bp,encoding 114 amino acids.The specific antigen gene was successfully amplified,SDS-PAGE showed that the recombinant protein p ET-Fh was expressed as an inclusion body with a clear band about 32 ku,and Western-blot demonstrated that the recombinant protein can be specifically recognized by F.hepatica positive sera.The results suggest that the hypothesized protein D915000758 can be used as a potential candidate antigen for the diagnosis of F.hepatica disease,and it should be used in the research of diagnosis technology for liver fluke disease caused by F.hepatica infection.