旋毛虫半胱氨酸蛋白酶抑制剂基因TsCystatin1的克隆及序列分析  

Cloning and sequence analysis of TsCystatin1 cystatin gene of Trichinella spiralis

作　　者：姚菊霞[1] 李文卉[1] 盖文燕[1] 曲自刚[1] 谢志宙[1] 王艳华[1] 张德林[1] 付宝权[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点开放实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2011年第6期569-574,共6页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2007BAD40B03);中央级公益性科研院所基本科研业务费专项(0032007012);甘肃省科技重大专项(0702NKDA039);教育部留学回国人员科研启动基金资助项目

摘　　要：为了研究旋毛虫半胱氨酸蛋白酶抑制剂的功能,应用电子克隆的方法从GenBank EST数据库获得1个旋毛虫半胱氨酸蛋白酶抑制剂基因(TsCystatin1)的部分cDNA序列,据此设计引物,并以旋毛虫新生幼虫总RNA为模板,进行反转录及PCR,克隆到该基因的完整开放阅读框序列,利用生物信息学软件对其进行了预测分析。结果表明,TsCystatin1 cDNA序列含有一个由666个核苷酸组成的开放阅读框架,编码由221个氨基酸残基组成的多肽,蛋白分子质量理论值为24.3ku,理论等电点为4.44。TsCystatin1第1～25位氨基酸残基为信号肽序列,具有半胱氨酸蛋白酶抑制剂的保守序列QVVAG,其C末端具有3处N-糖基化位点以及4个链内二硫键所需的半胱氨酸残基。该蛋白的二级结构中α螺旋占18.6%,β折叠占23.5%,其余57.9%为转角。结构域分析表明,该蛋白具有一个半胱氨酸蛋白酶抑制剂样结构域,属于半胱氨酸蛋白酶抑制剂家族2,与其他线虫半胱氨酸蛋白酶抑制剂的同源性较高。In order to study the function of cystatin of Trichinella spiralis,the cDNA sequence of cystatin of T.spiralis（TsCystatin1） was obtained using in silico cloning from GenBank database and RT-PCR from total RNA of newborn larvae.The open reading frame（ORF） was cloned and analyzed with bioinformatics software.The results indicated that the cDNA sequence contained an ORF of 666 nucleotides and the deduced protein consisted of 221 amino acids with the theoretical molecular weight of 24.3 ku and isoelectric point of 4.44.The signal peptide sequence of TsCystatin1 located between amino acids 1 and 25.The conserved QVVAG of cystatin,3 N-glycosylation sites and 2 disulfide bonds were also identified.Analysis of secondary structure revealed that α-helix,β-strand and loop were 18.6%,23.5% and 57.9%,respectively.Structural domain analysis indicated that the protein contained a cystatin-like domain belonging to the cystatin family and had high similarity to those of other nematodes.

关 键 词：旋毛虫 半胱氨酸蛋白酶抑制剂 克隆 序列分析 

分 类 号：S852.731[农业科学—基础兽医学]