[马立克氏病病毒Rispens株糖蛋白B基因的克隆及重组禽痘病毒的构建]

[Cloning of glycoprotein B gene from strain Rispens of Marek's disease virus and construction of recombinant fowlpox virus].

作者信息

Xing L, Peng D, Zhu A, Liu X, Zhang R

机构信息

Department of Veterinary Medicine, Agricultural College, Yangzhou University, Yangzhou 225009.

出版信息

Wei Sheng Wu Xue Bao. 1999 Apr;39(2):164-7.

PMID:12555423

Abstract

Purified DNAs from Chicken Embryo Fibroblast (CEF) cultures infected with MDV strain Rispens were used as templates. Specific fragment with the size of about 2.9 kb was successfully amplified through Polymerase Chain Reaction(PCR) and identified to be gB gene of MDV by dot blot hybridization with a digoxigenin-labelled MDV gB specific oligonucleotide probe. The gB gene from strain Rispens was cloned into pUC19 and FPV insertion vector pFG1175-1 to construct plasmid pMGB and pFGBR1775-1 respectively. DOSPER liposome-mediated transfection with insertion vector DNA pFGBR1175-1 was performed on CEF monolayers infected with FPV 3-4 h earlier. Recombinant FPV was clone purified. Immunofluorescence Assay(IFA) showed that MDV gB gene had been expressed in FPV.

摘要

以感染马立克氏病病毒（MDV）Rispens株的鸡胚成纤维细胞（CEF）培养物中的纯化DNA为模板。通过聚合酶链反应（PCR）成功扩增出大小约为2.9 kb的特异性片段，并用地高辛标记的MDV gB特异性寡核苷酸探针进行斑点杂交，鉴定为MDV的gB基因。将Rispens株的gB基因分别克隆到pUC19和禽痘病毒（FPV）插入载体pFG1175-1中，构建质粒pMGB和pFGBR1775-1。用插入载体DNA pFGBR1175-1进行DOSPER脂质体介导的转染，转染对象为3-4小时前感染FPV的CEF单层细胞。对重组FPV进行克隆纯化。免疫荧光分析（IFA）表明MDV gB基因已在FPV中表达。