猪圆环病毒2型感染性克隆的构建及拯救病毒的双抗体夹心ELISA鉴定  

Construction of porcine circovirus type 2 infectious clones and identification of rescued viruses using double-antibody sandwich ELISA

作　　者：贺紫琴 宁李纳 范杰 凌同 刘甜甜 李欣 陈志雄 黎满香[1] 葛猛[1] HE Ziqin;NING-LI Na;FAN Jie;LING Tong;LIU Tiantian;LI Xin;CHEN Zhixiong;LI Manxiang;GE Meng(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Engineering Technology Center,Hunan Sinoland Biological Pharmaceutical Co.,Ltd.,Changsha 410300,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南中岸生物药业有限公司工程技术中心,湖南长沙410300

出　　处：《畜牧与兽医》2024年第7期95-101,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目(32072871);湖南省重点研发计划项目(2023NK2017);湖南省普通高等学校科技创新团队支持计划项目。

摘　　要：旨在构建猪圆环病毒2型(PCV2)感染性克隆,并利用双抗体夹心ELISA对拯救病毒进行鉴定。通过PCR从阳性病料中扩增出PCV2两种基因型2b与2d的全基因组DNA,并分别克隆入pMD-19T载体,获得2个重组质粒,命名为p19T-PCV2b与p19T-PCV2d。利用SacⅡ酶切获得l 767 bp的PCV2全基因组,并在体外分别用T4连接酶连接而环化,将环状基因组转染至PK-15细胞,传至P6代,利用间接免疫荧光试验(IFA)对拯救病毒进行鉴定,结果表明PCV2b与PCV2d感染性克隆可拯救出病毒。利用PCV2单克隆抗体(单抗)作为捕获抗体,用辣根过氧化酶(HRP)标记的PCV2多抗作为检测抗体,建立检测PCV2病毒粒子的双抗体夹心ELISA方法,用于PCV2拯救病毒的鉴定。将该检测方法与IFA和半数组织细胞感染量(TCID_(50))结果进行对比分析,结果显示,ELISA检测值与Image-J所计算的IFA荧光强度及TCID_(50)的相关系数分别达到0.7和0.8以上,说明该方法与IFA及TCID_(50)检测结果均具有较强的一致性,可以用于PCV2拯救病毒的快速鉴定。The whole genome DNA of two genotypes,2b and 2d,of porcine circovirus type 2(PCV2)was amplified from the positive samples by PCR,and cloned into the pMD-19T vector,respectively.Then,the resulting recombinant plasmids were named p19T-PCV2b and p19T-PCV2d,respectively,and the 1767 bp whole genome of PCV2 was cut out by SacⅡ,and was cyclized by T4 ligase in vitro.The circular genome was transfected into PK-15 cells and passaged to the P6,and the rescued virus was identified by indirect immunofluorescence assay(IFA).The results showed that PCV2b and PCV2d infectious clones were rescued to generate the viruses.A double-antibody sandwich ELISA method for the detection of PCV2 virions was established by using PCV2 monoclonal antibody as the capture antibody and horseradish peroxidase(HRP)-labeled PCV2 polyclonal antibody as the detection antibody for the identification of PCV2 rescued virus.The above results indicated that the correlation coefficients between the ELISA detection value and the IFA fluorescence intensity and TCID_(50) value calculated by Image-J were above 0.7 and 0.8,respectively,suggesting that the ELISA method was in strong consistency with the IFA and TCID_(50) results,and could be used for the rapid identification of PCV2 rescued virus.

关 键 词：PCV2 感染性克隆 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]