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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
