摘要
目的寻找可用于日本血吸虫病诊断或疗效考核的抗原分子。方法在安徽省安庆市某现场,采集经粪检(Kato-Katz法,2送6检)日本血吸虫卵阳性的10份患者血样,患者年龄13～64岁,EPG为4～172;并收集该10例患者经吡喹酮(60 mg/kg×2 d)治疗6个月后的血样,将吡喹酮治疗前和治疗后的血清各10份分别混合,分别筛选虫卵cDNA文库,将所获阳性克隆分类,测定阳性克隆插入片段的DNA序列,经BLAST程序分析同源性,并利用在线的生物信息学软件预测阳性克隆基因编码蛋白的结构和功能。结果初次筛选共获得75个阳性克隆,其中46个阳性克隆吡喹酮治疗前和治疗后的日本血吸虫病患者血清反应一致(为Ⅰ类),21个阳性克隆吡喹酮治疗前的血清强于治疗后的(为Ⅱ类);8个阳性克隆吡喹酮治疗后的血清强于治疗前的(为Ⅲ类)。结合反应强度、分类和插入片段的大小选择了14个阳性克隆进行测序和生物信息学分析,多数阳性克隆插入片段均属于虫卵毛蚴抗原家族,1个阳性克隆的插入片段与含EF-hand结构域的钙结合蛋白具有一定的同源性(分值=143),1个阳性克隆的插入片段与应激反应组分1前体具有较高的同源性(分值=487),其余阳性克隆的插入片段仅与假定蛋白有一定的同源性,蛋白功能无明确注释。结论用吡喹酮治疗前后的日本血吸虫病患者血清筛选虫卵cDNA文库,获得的29个阳性克隆在吡喹酮治疗前后有差异。
Objective To obtain promising antigens for diagnosis or therapeutic efficacy assessment of schistosomiasis japonica.Methods At an endemic area in Anqing of Anhui Provinces,Kato-Katz method（six slides from two consecutive stool samples） was used to examine schistosome eggs and serum samples of 10 egg-positive patients were collected.The EPG of the ten patients,aged 13 to 64,was among 4 to 172.The patients were then treated with praziquantel 60 mg/kg for two days.Sera of the same patients were collected again at six months post treatment.The egg cDNA library was immunoscreened with the pre-and post-treatment serum samples,and the positive clones were classified according to different reactions.The inserted fragments of positive clones were sequenced and analyzed for their homology through BLAST program.The amino acid sequences of the proteins were deduced from the gene sequences,and their structures and functions were predicted by bioinformatics tools.Results For the first round,75 positive clones were screened from the egg cDNA library.Among them,21 clones showed stronger reaction with the pre-treatment sera,and 8 clones showed stronger reaction with the post-treatment sera.According to the intensity of the reaction,types and the length of inserted fragments,14 positive clones were selected for further study.Most of the clones belonged to miracidial antigen family,one clone was significantly homologous to the calcium-binding EF-hand,a domain-containing protein（score=143）,one was highly homologous to the cell wall integrity and stress response component 1 precursor（score=487）,and another four were homologous to the hypothetical protein.Conclusion Twenty nine positive clones screened from the egg cDNA library show different reactions with the sera of schistosome-infected patients,before and after praziquantel treatment.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2012年第2期109-115,共7页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家传染病科技重大专项(No.2009ZX1004-302)
国家高技术研究发展计划(863计划)项目(No.2007AA02Z153)
国际科技合作与交流专项(No.2010DFA33970)~~
关键词
日本血吸虫
虫卵
CDNA文库
免疫筛选
疗效考核
Schistosoma japonicum
Egg
cDNA library
Immunoscreening
Assessing therapeutic efficacy
