四种隐孢子虫半胱氨酸蛋白酶Cryptopain-1基因的克隆和序列分析  

CLONING AND SEQUENCE ANALYSIS OF CYSTEINE PROTEASE CRYPTOPAIN-1 GENE OF FOUR CRYPTOSPORIDIUM SPECIES

作　　者：王晓娟[1,2] 米荣升[2] 周鹏[2] 黄燕[2] 王向佩[1,2] 杨晓娇[2] 石凯[2] 刘宇轩[2] 雷晓思 陈兆国[2] 赵权[1] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室中国农业科学院动物源性食品安全研究中心,上海200241

出　　处：《中国动物传染病学报》2014年第5期41-48,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家科技重大专项项目(2012ZX10004220);中央级公益性科研院所基本科研业务费专项资金项目(2013JB13);家畜疫病病原生物学国家重点实验室开放基金课题(SKLVEB2013KFKT017);上海市科技兴农重点攻关项目(沪农科攻字[2005]第3-4号)

摘　　要：为克隆不同种隐孢子虫(Cryptosporidium spp.)的半胱氨酸蛋白酶Cryptopain-1基因,分析其核苷酸与编码氨基酸序列的变异情况,根据GenBank上公布的微小隐孢子虫(C.parvum)Cryptopain-1基因序列设计合成引物,用PCR技术从不同种隐孢子虫基因组DNA中扩增Cryptopain-1基因,并将其克隆至pZeroBack/blunt载体,阳性克隆经PCR鉴定正确后测序,与微小隐孢子虫Cryptopain-1基因进行序列同源性比对,并进行系统进化分析。结果显示,本研究成功从泰泽隐孢子虫(C.tyzzeri)、火鸡隐孢子虫(C.meleagridis)、兔隐孢子虫(C.cuniculus)基因组DNA中扩增出Cryptopain-1基因。与微小隐孢子虫Cryptopain-1基因比较,泰泽隐孢子虫、火鸡隐孢子虫、兔隐孢子虫Cryptopain-1基因核苷酸序列相似性分别为98.67%、94.53%、98.34%,演绎的氨基酸序列相似性分别为99.00%、96.51%、99.00%。研究结果为利用Cryptopain-1基因进行隐孢子虫的代谢、致病机理等研究奠定了基础。In order to clone Cryptopain-1 gene of different Cryptosporidium species for analysis of genetic variation, a pair of specific primers were designed based on the sequence of C parvum Cryptopain-1 gene published on GenBank. The Cryptopain-1 genes of different Cryptosporidium species were amplified from their genomes using PCR technique and then cloned into pZeroBack/blunt vectors. The cloned genes from the positive recombinant plasmids were sequenced and phylogenetically analyzed. Cryptopain-1 gene of C parvum shared nucleotide identities at 98.67%, 94.53% and 98.34% with C tyzzeri, C meleagridis and C cuniculus. The information obtained from the present study will be useful for research on metabolism, pathogenecity of Ctyptospotieium.

关 键 词：隐孢子虫 Cryptopain-1基因 克隆 序列分析 

分 类 号：S852.723[农业科学—基础兽医学]