褐黄血蜱cystatin基因全长的克隆及原核表达研究  

Cloning and prokaryotic expression of full-length cystatin gene from Haemaphysalis flava

作　　者：和小明[1] 刘磊[1] HE Xiaoming;LIU Lei(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院

出　　处：《黑龙江畜牧兽医》2018年第23期13-16,20,265,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31372431)

摘　　要：为了探讨褐黄血蜱体内半胱氨酸蛋白抑制剂(cystatin)蛋白及其编码基因的情况,试验基于褐黄血蜱唾液腺转录组数据库中搜索的注释为cystatin的contig_43533序列设计引物,RACE扩增其5′端和3′端,拼接获得cDNA全长,利用生物信息学软件进行分析;以饱血褐黄血蜱雌成虫全蜱总cDNA为模板,扩增cystatin-ORF,构建重组表达载体,原核表达、纯化制备cystatin重组蛋白。结果表明:褐黄血蜱cystatin cDNA全长635 bp,包含396 bp的开放阅读框架,编码一个含131个氨基酸残基的蛋白,原核表达可获得褐黄血蜱cystatin重组蛋白。说明褐黄血蜱cystatin蛋白能在大肠杆菌内以可溶性形式稳定表达。The aim of the present study was to investigate the cysteine protease inhibitor ( cystatin) protein and its encoding gene in Haemaphysalis flava. The primers were designed based on the contig_43533 sequence ( annotated as a cystatin) in the salivary glands transcriptome database of Haemaphysalis flava,and the 5 ' and 3' ends were amplified by RACE to obtain the full length of cDNA,which was analyzed by bioinformatics software. The recombinant expression vector was constructed by amplification of cystatin-ORF from the total cDNA of Haemaphysalis flava,and the recombinant protein of cystatin was prepared by prokaryotic expression and purification. The results showed that the full-length cystatin cDNA of Haemaphysalis flava was 635 bp,which contained 396 bp ORF,encoding a protein containing 131 amino acid residues. The recombinant protein of Haemaphysalis flava cystatin was obtained by prokaryotic expression. The results indicated that the cystatin protein of Haemaphysalis flava could be expressed steadily in a soluble form in Escherichia coli.

关 键 词：褐黄血蜱 半胱氨酸蛋白酶抑制剂 基因全长 克隆 原核表达 纯化 

分 类 号：S855.99[农业科学—临床兽医学] Q785[农业科学—兽医学]