棘阿米巴表达文库的免疫学筛选  

Immunological screening of cDNA expression library from Acanthaoeba

作　　者：张宏梅[1] 郑文彧[2] 刘迪[1] 时文艳[1] 孙宏宇[1] 崔佰吉[1] 高俊涛[1] 冯宪敏[1] ZHANG Hong-mei;ZHENG Wen-yu;LIU Di;SHI Wen-yan;SUN Hong-yu;CUI Bai-ji;GAO Jun-tao;FENG Xian-min(Department of Pathogenic biology, Jilin Medicin University, Jilin 132013, China;Department of Hand Microsurgery, Central Hospital of Jilin City, Jilin 132000, China)

机构地区：[1]吉林医药学院病原生物学教研室,吉林吉林132013 [2]吉林市中心医院,吉林吉林132000

出　　处：《中国兽医杂志》2018年第1期17-20,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31572262);吉林省科技厅国际合作项目(20160414050GH)

摘　　要：通过棘阿米巴感染兔血清对棘阿米巴原虫全长cDNA文库进行免疫学筛选,采用角膜注射的方法构建棘阿米巴感染兔模型,收集感染后第1、7、14、21、28、35、42天兔血清,ELISA法测定抗体滴度;选用感染后第7、14、21天和28天混合血清对c DNA文库进行免疫学初筛和复筛;阳性克隆送样测序,并进行PCR鉴定和序列分析。结果:角膜刮片镜检,感染动物试验组均检出棘阿米巴的包囊和滋养体,感染血清抗体滴度在感染后第7天升高显著,在感染后第28天达到高峰;经免疫学筛选共获得3个阳性克隆,经测序和PCR鉴定,3个克隆的插入序列和ORF长度分别为:actin1,774 bp、polyubiquitin,234 bp和HSP20,564 bp。本研究为进一步棘阿米巴原虫感染的快速诊断试剂和免疫预防分子的筛选奠定基础。The whole c DNA Library of Acanthaoeba protozoan was screened with the method of immunological screening. The rabbit model of Acanthamoeba keratitis was constructed with method of injection infection. Anti-Acanthamoeba antibodies in sera were determined before infection and on different time postinfection. The rabbit models of Acanthamoeba keratitis were built successfully and proved by corneal scraping examination. Antibody titer rose continuously with time after infection and reached the highest levels on 28 th days. After immunological screening,3 positive clones were obtained. After sequencing and PCR identification,the insertion sequences（ ORF length） of 3 clones were actin1（ 774 bp）,polyubiquitin（ 234 bp） and HSP20（ 564 bp）,respectively. This study lays a foundation for further screening of Acanthamoeba infection,rapid diagnostic reagents and immunization molecules.

关 键 词：棘阿米巴原虫 棘阿米巴角膜炎 CDNA文库 免疫学筛选 

分 类 号：S855.91[农业科学—临床兽医学]