微小隐孢子虫cDNA文库的构建与免疫学筛选  

Construction and immunoscreening of cDNA library of Cryptosporidium parvum

作　　者：高依然[1] 张静[1] 韩福松[1] 陆慧君[1] 姜宁[1] 陈启军[1] 尹继刚[1] 

机构地区：[1]吉林大学人兽共患病研究所,人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《西北农林科技大学学报（自然科学版）》2013年第8期7-12,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"十一五"传染病重大专项(2008ZX10004-011);国家自然科学基金项目(31025025;31272549)

摘　　要：【目的】构建微小隐孢子虫cDNA文库,以筛选出可替代天然抗原用于诊断的重组抗原。【方法】首先纯化微小隐孢子虫卵囊,用Trizol Reagent提取总RNA,构建全长cDNA;分别用蛋白酶K与Sfi消化、酶切处理cDNA,采用Column Spin H-1000分离柱分离cDNA,连接λTriplEx2载体,构建cDNA文库。扩增cDNA文库,用兔抗微小隐孢子虫血清作探针对cDNA文库进行初筛和复筛,将复筛得到的阳性克隆连接PMD18-T载体,测序后进行序列比对。【结果】微小隐孢子虫cDNA文库噬菌斑插入片段长度为1.5kb左右,库容大于107。经过筛选共获得了20个微小隐孢子虫阳性克隆,测序后从中选出了4个主要免疫识别抗原蛋白,分别为MUCIN蛋白、乳酸脱氢酶、微管相关蛋白和子孢子抗原蛋白。【结论】成功构建了微小隐孢子虫cDNA文库,并筛选出了4个主要免疫识别抗原蛋白,为隐孢子虫的血清学诊断提供了重组抗原。A cDNA expression library of Cryptosporidium parvum was constructed to select recombinant antigens which would replace the natural antigens for diagnosis of cryptosporidiosis.【Method】Cryptosporidium parvum oocysts were purified by percoll centrifugation.Total RNA was extracted with Trizol Reagent for constructing cDNA with full-length.cDNA was digested with proteinase K and Sfi and separated with Column Spin H-1000.cDNA was ligated into λTriplEx2 vector to construct the expression library.The library was amplified and screened with rabbit anti-Cryptosporidium parvum serum.Inserted fragments from positive clones were connected to PMD 18-T and sequenced for analysis.【Result】The mean size of the inserted cDNA fragments was 1.5kb,the capacity of the library was greater than 10 7,and 20 positive clones of Cryptosporidium parvum were identified by immunological screening.Four gene sequences,including MUCIN protein,Lactate dehydrogenase,Microtubule-associated protein and Sporozoite antigen protein,were identified.【Conclusion】Cryptosporidium parvum cDNA expression library was successfully constructed and screened,and four candidate immunodominant proteins were identified which would be used for diagnosis of cryptosporidiosis.

关 键 词：微小隐孢子虫 CDNA文库构建 免疫学筛选 

分 类 号：S852.723[农业科学—基础兽医学]