阳性水牛、黄牛IgG筛选日本血吸虫成虫噬菌体展示cDNA文库

摘要

为寻找有效的家畜日本血吸虫病诊断抗原,从感染日本血吸虫的水牛、黄牛血清中纯化IgG,以亲和淘洗方法筛选日本血吸虫成虫(44日龄)噬菌体展示cDNA文库.经每种动物IgG三轮(总计六轮)筛选后,随机挑取53个阳性噬菌体克隆进行序列测定及生物信息学分析.获得了9个EST序列,其中7个EST序列与已知基因或表达序列标签同源,2个EST序列与已知基因或表达序列标签均无同源性,以噬菌体展示载体的10B衣壳蛋白开放阅读框(open reading frame,ORF)为依据,对上述7个与GenBank数据库资料同源的EST进行对码分析,结果7个EST序列能和噬菌体展示载体阅读框对码,为有效展示序列.其中3个EST具有较高的重复度,在51个有效克隆中,25个为HSP70 homolog基因,15个为Keratin9基因有重复,6个为Ornithine--oxo-acid transaminase基因.研究结果显示,HSP70 homolog基因、Keratin9基因和Ornithine--oxo-acid transaminase基因作为诊断家畜血吸虫病候选抗原基因,值得进一步开展克隆、表达和诊断效果评估方面的研究.%In order to find the effective diagnostic antigen for S.japonicum infection in domestic animals, IgG preparations were purified from serum samples of infected buffalo and cattle and used to screen a phage display cDNA library from 44-day-old worms. After a total of six rounds of biopanning, 53 positive clones were randomly picked and sequenced and 9 ESTs including 2 unnamed ESTs were obtained. With open reading frame (ORF) of 10B capsid protein in phage display vector as the basis, 7 ESTs were determined to be effective display sequences. Among the 7 effectively displayed ESTs, 3 ESTs had higher multiplicity. Among the 51 positive clones sequenced, 25 clones were HSP70 homolog, 15 clones were Keratin 9 and 6 clones were Ornithine-oxo-acid transaminase. These results obtained here suggested that the genes encoding HSP70 homolog, Keratin 9 and Ornithine-oxo-acid transaminase might be relevant to the candidate diagnostic antigens for detection of S.japonicum infection in domestic animals.