猪圆环病毒2型Cap蛋白特异性抗原表位重组腺病毒的构建及鉴定  

Construction and Indentification of Recombinant Adenoviruses Containing the ORF2 Gene of Porcine Circovirus 2

作　　者：郭全海[1,2,3] 陈陆[1] 王川庆[1] 吴德峰[3] 王永生[1] 李志娟[1] 王艳[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]商丘职业技术学院,河南商丘476000 [3]福建农林大学,褔建福州350002

出　　处：《中国兽医学报》2011年第8期1099-1102,1110,共5页Chinese Journal of Veterinary Science

基　　金：国家高技术研究与发展计划项目(2007AA100606);河南省高等学校青年骨干教师计划资助项目(2010GGJS-045)

摘　　要：猪圆环病毒2型PCV2ORF2基因表达的Cap蛋白含有病毒的主要抗原表位,是PCV2基因工程苗研究的主要候选蛋白。根据GenBank公布的PCV2基因序列设计1对PCR引物,从感染有PCV2的PK-15细胞中扩增出包含2个特异性表位的Cap蛋白基因片段。把Cap蛋白基因目的基因克隆入腺病毒5型穿梭质粒中,构成重组穿梭质粒rpShuttle-CMV-276。分2步把骨架质粒pAdeasy-1和重组穿梭质粒rpShuttle-CMV-276分别用化学方法转化入BJ5183感受态工程菌中,同源重组后的质粒转染HEK-293A细胞,在细胞中包装出重组腺病毒。通过PCR、IPMA、免疫荧光及ELISA检测证明PCV2ORF2基因在HEK-293A中获得了表达。为PCV2的快速检测方法(检测试纸条)的建立及腺病毒活载体基因工程苗的研究奠定基础。Expressing protein contains govering antigeon epitopes of porcine circovirus serotype 2 gene,which is reaschered widely for genetic engineering vaccine.Aaccording to Genbank,designed a pair of primers which can amplify Cap protein gene contained two specific epitopes from PK-15 affected PCV2 by PCR.The gene was subcloned into the shuttle vector pShuttle-CMV of adenoviruses serotype 5 constructed recombinant shuttle plasmid rpShuttle-CMV-276.Backbone vector pAdeasy-1 and rpShuttle-CMV-276 transformed into competent BJ5183 by chemical method differently,they were homologous recombinated in it.At last,produced recombination adenoviruses after they ransformed into HEK-293A cell.Expression of ORF2 gene protein in was HEK-293A cell detected by PCR、IPMA、immunofluorescent assy and ELISA.The study laid the foundation for rapid detection method（detection dipstick） and development of the recombinant adenoviruses vaccine against PCV2.

关 键 词：猪圆环病毒2型(PCV2) Cap蛋白基因(ORF2基因) 腺病毒 鉴定 

分 类 号：S852.65[农业科学—基础兽医学]