摘要
扩增鸭瘟病毒(DPV)生长非必需区的TK基因,并在其中间引入Bgl II酶切位点,再之克隆到pUC19载体,获得载体pTK。用限制性内切酶从已有质粒pcDNA-LacZ上切下CMV启动子、多克隆位点、SV40及LacZ的完整的基因表达盒,插入到pTK的TK基因中,获得质粒pTCL。用质粒T-VP1做模板,扩增出I型鸭甲肝病毒(以前称为血清I型鸭肝炎病毒)VP1基因,克隆到质粒pTCL表达盒的多克隆位点KpnI与XbaI之间,构建含LacZ及VP1基因的转移载体质粒pT-CL-VP1。将此转移载体与鸭瘟病毒C-KCE毒株共转染鸡胚成纤维细胞(CEF),经蓝斑克隆筛选和纯化,获得了遗传性状稳定的表达I型鸭甲肝病毒VP1基因的重组鸭瘟病毒。
The TK gene （about 2.5kb）, the nonessential replication for duck plague virus （DPV） was amplified by PCR and then cloned into pUC19 vector to obtain pTK vector. According to the restriction endonulease recognized sites in the vector pcDNA-LacZ, the CMV promoter, multiple cloning sites, SV40, and the complete LacZ gene expression cassette were obtained by means of digestion with restriction enzyme and then inserted into middle of the TK gene, obtaining plasmid pTCL.Using the primers synthesized according to the type I duck hepatitis virus VP1 gene sequence published in Genebank, the VP1 gene was amplified from the plasmid T-VP1 as template and cloned into between the the polyclonal site KpnI and XbaI of plasmid pTCL, thus obtaining the pTCL-VP1 plasmid with LacZ and VP1 gene.The constructed transfer vector was cotransfeeted on CEF cells with the C-KCE strains, then to obtain the pure VPl-fowlpox virus recombinant by clonning and purification of blue plaque.
出处
《中国动物检疫》
CAS
2010年第6期28-31,共4页
China Animal Health Inspection
基金
863项目的家禽重要病毒病基因工程疫苗研究和创新(2006AA10A205)
