山羊痘病毒SS株5个ANK基因缺失的重组病毒构建  

Construction of recombinant virus with deletion of 5 ANK genes of goat pox virus SS strain

作　　者：杨勇飞 张成 张雪萍 童剑军 高娜 李有文[1,2] Yang Yongfei;Zhang Cheng;Zhang Xueping;Tong Jianjun;Gao Na;Li Youwen(College of Animal Science,Tarim University,Xinjiang Alaer 843300;Key Laboratory of Tarim Animal Husbandry Science and Technology Corps,Xinjiang Production&Construction Corps,Xinjiang Alaer 843300;College of Life Sciences,Tarim University,Xinjiang Alaer 843300)

机构地区：[1]塔里木大学动物科学学院,新疆阿拉尔843300 [2]新疆生产建设兵团塔里木畜牧科技兵团重点实验室,新疆阿拉尔843300 [3]塔里木大学生命科学学院,新疆阿拉尔843300

出　　处：《现代畜牧兽医》2021年第1期58-62,共5页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：国家自然科学基金项目(31760741、31360616)。

摘　　要：为研究锚定蛋白基因(Ankyrin,ANK)对山羊痘病毒的影响,试验采用融合PCR和Overlap PCR技术扩增山羊痘病毒SS株5个ANK基因(ANK010、ANK138、ANK140、ANK141.2和ANK145)两端侧翼序列和绿色荧光蛋白(GFP)基因,并将其产物连接Trans1-T 1载体构建ANK缺失的转移载体,经过菌液PCR以及质粒双酶切鉴定,阳性重组质粒用Lip 2000转染至已经感染羊痘病毒SS株的羊睾丸原代细胞,依报告基因GFP的表达情况在荧光显微镜下筛选目的基因缺失的重组病毒,同时设立不感染病毒的对照。结果表明:通过融合PCR方法成功扩增山羊痘病毒SS株ANK基因010和138的两端侧翼序列及GFP基因片段,大小约1200 bp;通过Overlap PCR方法成功得到ANK基因140、141.2和145基因的侧翼及GFP基因片段,大小约900 bp,与理论相符。研究成功构建了基因缺失转移载体,将其转染感染SS病毒的细胞中,5个ANK基因缺失的表达载体均可见绿色荧光斑点,说明得到各自基因缺失的重组羊痘病毒。In order to investigate the effect of ankyrin(ANK)gene on goatpox virus,the flanking sequences and green fluorescent protein(GFP)genes at both ends of 5 ANK genes(ANK010,ANK138,ANK140,ANK141.2 and ANK145)of goatpox virus SS strain were amplified using fusion PCR and Overlap PCR,and the products were linked to vector Trans1-T 1 to construct the transfer vector of each ANK deletion.After identification by PCR of bacterial liquid and double enzyme digestion of plasmid,the positive recombinant plasmid was transfected into primary goat testis cells infected with goatpox virus SS strain by Lip 2000.According to the expression of GFP reporter gene,the recombinant virus with deletion of target genes was screened under a fluorescence microscope.The results showed that the flanking sequences and GFP gene fragments at both ends of 5 ANK genes of goatpox virus SS strain were successfully amplified in the study,with a size of about 1200 bp,which were successfully inserted into Trans1-T 1 to construct the transfer vector with gene deletion.After transfection,the recombinant virus with deletion of 5 ANK genes were obtained from virus-infected cells.In addition,non-virus-infected controls were made.The results showed that the flanking sequences and GFP fragments at both ends of ANK010 and ANK138 of goatpox virus SS strain were successfully amplified by fusion PCR,with a length of about 1200 bp,and the flanking sequences and GFP fragments of ANK140,ANK141.2 and ANK 145 were successfully obtained by Overlap PCR,with a length of about 900 bp,which were consistent with the theoretical lengths.Additionally,transfer vectors with gene deletion were successfully constructed,and transfected into SS virus-infected cells.Compared with controls,green fluorescent spots were observed in all the expression vectors with deletion of the 5 ANK genes,suggesting that the recombinant goatpox viruses with deletion of each gene were obtained.

关 键 词：羊痘病毒 GFP 基因缺失 融合PCR 重组病毒 Overlap PCR 

分 类 号：S855.3[农业科学—临床兽医学]