摘要
应用PCR从pMD18-T-E0质粒中扩增编码CSFV E0蛋白的基因片段,定向克隆到重组腺病毒Adeasy-1系统的穿梭质粒pAdTrack-CMV上,采用细菌内同源重组“两步转化法”构建携带CSFV E0基因的重组腺病毒基因组质粒pAdEasy-E0,转染293细胞,成功包装出重组腺病毒pAd-E0,PCR证实E0基因已整合至腺病毒基因组中,用Western blot检测到重组病毒感染293细胞中E0蛋白的表达。重组病毒免疫小鼠和猪,结果2次免疫后产生明显的免疫应答,ELISA检测小鼠血清抗体滴度分别为1∶512和1∶10240;猪血清抗体滴度分别为1∶16和1∶64。本研究成功构建了表达猪瘟病毒E0基因的非复制型重组腺病毒,该重组病毒免疫小鼠可产生较高的抗体滴度,免疫猪后能提供一定的保护效果。
Classical swine fever virus （CSFV）E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the adenoviral shuttle plasmid pAdTrack-CMV. A two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAdEasy-E0. The resultant recombinant DNA was transfected into 293 cells to yield packaged adenovirus. E0 gene was confirmed to be integrated into the genome of recombinant adenovirus by PCR, and the expression of E0 was detected in the 293 cells infected with recombinant adenovirus by Western blot. ELISA results showed that systematical immune responses to CSFV virus could be induced effectively in mice after twice immunizations through intraperitoneal route. The replication-defective recombinant adenovirus expressing （CSFV） E0 was constructed and could elicit favorable immune responses in mice,and offered protection for pigs to CSFV.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第5期482-487,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
陕西省重大科技攻关项目(2006kz07-G2)
国家自然科学基金项日(30471290)
关键词
腺病毒
猪瘟病毒
E0基因
同源重组
adenovirus
classical swine fever virus
E0 gene
homogenous recombination
