绵羊痘病毒甘肃古浪株RING finger基因的克隆表达及序列分析  

Cloning,expression and sequence analysis of RING finger gene of sheeppox virus Gansu Gulang strain

作　　者：陈晓倩 吴国华[2] 郭小腊 杨静[2] 吴金恩 颜鲁军 周雪雁[1] 郑亚东[2] 陈轶霞[1] CHEN Xiao-qian;WU Guo-hua;GUO Xiao-la;YANG Jing;WU Jin-en;YAN Lu-jun;ZHOU Xue-yan;ZHENG Ya-dong;CHEN Yi-xia(College of Life Science and Engineering,Northwest University for Nationalities,Lanzhou 730030,China;State Key Laboratory of Veterinary Etiological Biology,Key Laboratory of Veterinary Parasitology of Gansu Province,Lanzhou Veterinary Research Institute,CAAS,Lanzhou 730046,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]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,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2019年第3期1-8,共8页Journal of Gansu Agricultural University

基　　金：国家自然科学基金项目(31560697);中央高校基本科研业务费创新团队培育项目(31920190027)

摘　　要：【目的】对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.【方法】以绵羊痘病毒甘肃古浪株DNA为模板,通过PCR扩增 RING finger 基因.利用Pfam数据库、DNAstar等软件进行序列及遗传进化分析;将 RING finger 基因片段插入原核表达载体pGEX-4T-1中,构建pGEX-SPPVRFP重组表达载体,在大肠杆菌BL21(DE3)中诱导表达,并进行SDS-PAGA和Western-blot分析.【结果】绵羊痘病毒 RING finger 基因由723个核苷酸组成,编码的240个氨基酸的分子量约为28.5 ku,具有RING finger结构域.不同羊痘病毒株间 RING finger 基因核苷酸序列同源性高达99.2%,氨基酸序列同源性高达98.3%.不同的RING finger蛋白都含有8个保守的半胱氨酸和组氨酸.SDS-PAGA分析显示,重组SPPVRFP大小约为55 ku,Western-blot分析显示,重组SPPVRFP不能与绵羊痘病毒阳性血清反应.【结论】成功克隆、表达并纯化了绵羊痘病毒 RING finger 基因,对SPPV GS-GL株RING-finger蛋白进行序列分析,推测其可能具有E3泛素连接酶活性.【Objective】 The gene of RING finger protein of sheeppox virus was cloned,expressed and sequenced,aiming to analyze whether sheeppox virus RING finger protein has E3 ubiquitin ligase activity in order to provide a foundation for further studies on the regulatory functions of SPPVRFP in the ubiquitin protease system during sheeppox virus infection.【Method】 The RING finger gene was amplified with PCR by using DNA of sheeppox virus Gansu Gulang strain as a template.The sequence and genetic evolution analysis were carried out by using Pfam database and DNAstar software,respectively.The RING finger gene fragment was ligated into the prokaryotic expression vector pGEX-4T-1,then the recombinant prokaryotic expression vector pGEX-SPPVRFP was constructed to induce the expression in E.coli BL21(DE3),and analyzed by SDS-PAGA and Western-blot.【Result】 The sheeppox virus RING finger gene consisted of 723 nucleotides,encoding 240 amino acids with a molecular weight mass of 28.5 ku,and it had a RING finger domain.The nucleotide sequence homology of RING finger genes among different sheeppox virus strains was as high as 99.2%,and the amino acid sequence homology was as high as 98.3%.It was found that the RING finger of SPPV had 8 conserved cysteines and histidines.The SDS-PAGA analysis showed that the relative molecular weight of recombinant SPPVRFP was about 55 ku,and Western-blot analysis showed that recombinant SPPVRFP did not reacted with the sheeppox virus positive sera.【Conclusion】 The sheeppox virus RING finger gene is successfully cloned,expressed and purified.It is speculated that it may have ubiquitin ligase acticity.

关 键 词：绵羊痘病毒 RING finger蛋白 序列分析 原核表达 

分 类 号：S852.654[农业科学—基础兽医学]