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

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 Cryptosporieium.