猪圆环病毒2型ORF-2全基因重组腺病毒的构建与鉴定  

Construction and Identification of Recombinant Adenovirus Containing the ORF-2 Complete Gene of Type 2 Porcine Circovirus

作　　者：林祥超 高志强[2] 张鹤晓[2] 张康 郭金玉[1] 牛建蕊[4] 张乐萃[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]北京出入境检验检疫局,北京通州100026 [3]北京森康生物技术开发有限公司,北京怀柔101400 [4]中国农业科学院兰州畜牧与兽药研究所,甘肃兰州730050

出　　处：《中国兽医杂志》2016年第1期24-26,共3页Chinese Journal of Veterinary Medicine

基　　金：质检公益性行业科研项目(201310253)

摘　　要：扩增猪圆环病毒2型的ORF-2全基因(702 bp),将目的片段与缺陷型腺病毒穿梭质粒pacAd5CMV K-NpA连接,构建重组穿梭质粒CMV-ORF-2。将穿梭质粒与骨架质粒pacAd5 9.2-100经限制性内切酶PacI线性化后共转染293T细胞,进行细胞内重组。7~15 d后获重组腺病毒rAd5 CMV-OPFF-2与对照组重组腺病毒rAd5 CMV-GFP。使用293细胞进行病毒增殖,对增殖稳定的rAd5 CMV-ORF-2进行鉴定;rAd5 CMV-OPFF-2 TCID_(50),为10^(7.5/)50μL,用该代次rAd5 CMV-ORF-2进行PCV-2阳性血清中和试验,结果表明,重组腺病毒成功,PCV-2阳性血清不能中和重组腺病毒。The 702 bp ORF 2 of PCV-2 was amplified and cloned into the defective adenovirus shuttle plasmid pacAd5 CMV K-NpA to construct recombinant shuttle plasmid CMV-ORF2.The shuttle plasmid and backbone plasmid pacAd5 9.2-100 were linearized using the restriction enzyme Pae I, and were then cotransfect into 293T cells. The ORF2 complete genome recombinant adenovirus rAd5 PCV-2 and the control CMV-GFP recombinant adenovirus rAd5 were obtained 7 to 15 days later. The recombi- nant viruses were propagated and identified in 293 cells. The recombinant viruses were stable in propagation and the TCID50 of tad5 CMV-ORF2 was 1075/50 μL. The PCV-2 positive sera and rAd5 CMV-ORF2 were used in neutralization test. The results showed that the construction succeeded and PCV-2 positive sera could not neutralize the recombinant adenovirus.

关 键 词：猪圆环病毒2型 ORF-2 缺陷型重组腺病毒 

分 类 号：S852.651[农业科学—基础兽医学]