马立克氏病病毒(MDV)糖蛋白gB在重组鸡痘病毒中的表达

Expression of Glycoprotein B from Marek's Disease Virus in Recombinant Fowlpox Virus

将马立克氏病病毒gB基因用EcoRⅠ从质粒pUR MDVEgB中切下。两端补平后,插入到质粒pEFgpt12s的PstⅠ酶切位点。构建了含有完整的MDVgB基因的转移载体pEFgpt12s gB。这个载体的特点是,它含有两个启动子,在强的复合启动子Spromoter的下游是插入的gB基因,另一个反向启动子vvP7 5的下游是标记基因Ecogpt,它的两端是FPV的非必需区。携带gB基因的质粒pEFgpt12s gB通过磷酸钙的方法转染用282E4株FPV感染3 4h的鸡胚成纤维细胞。通过药物筛选,得到含有gB基因的重组病毒。通过PCR、免疫荧光检测,证实了重组病毒中含有gB基因,并且gB糖蛋白也得到了表达。

B gene of Mareks disease virus was digested with EcoR1 from plasmid pURMDVEgB,and made it blunt.It was inserted into endonuclease Pst1 of plasmid pEFgpt12s.Then recombinant plasmid pEFgpt12sgB containing a whole MDVgB gene was synthesized.It was analyzed,the result suggested that it contains two promoters.The inserted B gene is at the downstream of the strong synthetic Spromoter,and the marked Ecogpt gene is under the control of the other reverse P75 promoter.Its two sides are nonessential region of FPV.Plasmid pEFgpt12sgB was transfected CEF which was infected with FPV for about 34 h through calcium phosohateDNA coprecipitation.Recombinant fowlpox virus containing gB gene was seleted by mycophenolic acid selection.Polymerase Chain Reaction(PCR) and Immunofluorescence Assay (IFA) showed that gB gene was in recombinant virus,and glycoprotein B was also expressed.