猪繁殖与呼吸综合征病毒ORF4/ORF5基因区间的反向遗传操作:插入外源序列特性研究  

REVERSE GENETIC MANIPULATION ON A FULL-LENGTH CDNA CLONE OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS:CHARACTERIZATION OF THE FOREIGN SEQUENCES INSERTED INTO THE SPACER OF ORF4/ORF5

作　　者：丛雁方[1] 张可煜[2] 高飞[1] 韦祖樟[1] 郑浩[1] 童光志[1] 袁世山[1] 郑海红[1] 

机构地区：[1]中国农业科学院上海兽医研究所动物传染病研究室,上海200232 [2]中国农业科学院上海兽医研究所动物药学研究室,上海200232

出　　处：《中国动物传染病学报》2012年第6期16-22,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(30972204)

摘　　要：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)ORF4/ORF5基因间隔区有10个核苷酸(nucleotide,nt),为了探索ORF4/ORF5基因间隔区作为外源基因插入靶点的可行性,本研究通过突变PCR技术,获得一系列ORF4/ORF5间在不同位置插入不同碱基数及特定序列的突变体,随后进行了病毒拯救和复制分析。结果表明,在ORF4/5的10 nts间隔序列之前至少可插入13 nts;在间隔序列之后只能允许3 nts的插入;间隔序列之间能允许至少18 nts的插入,但允许插入的序列具有特异性,由此,暗示ORF4/5允许插入的外源序列与插入位置有关。本研究获得的诸多突变病毒为进一步研制PRRSV基因标识疫苗奠定了基础。A spacer structure consisting of 10 nucleotides is located between ORF4 and ORF5 of porcine reproductive and respiratory syndrome virus（PRRSV）.In order to study the feasibility of the foreign sequences inserted between ORF4 and ORF5,a series of mutants were constructed from an infectious clone of pAPRRS and then a variety of nucleotide sequences were inserted into different positions in the spacer.The result showed that the spacer could tolerate more than 13 nucleotides if the insertion was imposed behind the stop codon of ORF4.Otherwise,only 3 nucleotides were permitted before the start codon of ORF5.However,as many as 18 nucleotides could be inserted within the spacer.These results indicated that the lengths of the inserted foreign sequences were related to specific positions within the spacer.In addition,the mutant viruses obtained from the present study can be used in the future for development of PRRSV marker vaccines.

关 键 词：猪繁殖与呼吸综合征病毒 ORF4 ORF5基因间隔区 外源序列 病毒感染性 

分 类 号：S852.659.6[农业科学—基础兽医学]