CAJ | 학술논문

为了构建鸭瘟鸡胚化弱毒株DEV(atten)的细菌人工染色体(BAC),将转移载体质粒pDEVgC-pHA2 DNA和DEV(atten) DNA共转染鸡胚成纤维细胞(CEF)进行重组,通过三轮挑斑纯化,以获得纯化的鸭瘟重组病毒。将纯化的鸭瘟重组病毒的DNA电转化到E. Coli DH10B中,对获得的克隆用酶切、PCR方法进行鉴定并测序。提取阳性克隆的DNA转染CEF,以拯救重组病毒。再通过将带有相同同源臂和UL44(糖蛋白质C,gC)基因的片段和阳性克隆的DNA共转染鸡胚成纤维细胞进行重组,以获得gC恢复病毒。采用0.01感染复数( MOI)感染CEF对亲本病毒、恢复病毒进行多步法生长曲线测定,比较其有无差异。结果显示：成功获得了DEV的mini-F重组病毒,命名为DEV(atten)-mini-F,获得2个阳性克隆S1、S2,将S1命名为pDEV(atten),应用pDEV(atten)拯救重组病毒获得gC恢复病毒DEV( atten)△gC-R,生长特性的结果显示亲本病毒与恢复病毒间无显著性差异。表明成功构建的DEV( atten)的细菌人工染色体为全基因组的感染性克隆。
위료구건압온계배화약독주DEV(atten)적세균인공염색체(BAC),장전이재체질립pDEVgC-pHA2 DNA화DEV(atten) DNA공전염계배성섬유세포(CEF)진행중조,통과삼륜도반순화,이획득순화적압온중조병독。장순화적압온중조병독적DNA전전화도E. Coli DH10B중,대획득적극륭용매절、PCR방법진행감정병측서。제취양성극륭적DNA전염CEF,이증구중조병독。재통과장대유상동동원비화UL44(당단백질C,gC)기인적편단화양성극륭적DNA공전염계배성섬유세포진행중조,이획득gC회복병독。채용0.01감염복수( MOI)감염CEF대친본병독、회복병독진행다보법생장곡선측정,비교기유무차이。결과현시：성공획득료DEV적mini-F중조병독,명명위DEV(atten)-mini-F,획득2개양성극륭S1、S2,장S1명명위pDEV(atten),응용pDEV(atten)증구중조병독획득gC회복병독DEV( atten)△gC-R,생장특성적결과현시친본병독여회복병독간무현저성차이。표명성공구건적DEV( atten)적세균인공염색체위전기인조적감염성극륭。
To construct a bacterial artificial chromosome of duck enteritis virus attenuated strain, DNA of transfer vector plasmid pDEVgC-pHA2 and duck enteritis virus( DEV) ( atten) strain were co-transfected into primary chicken em-bryo fibroblasts( CEFs) for recombination. Purified recombinant virus was achieved after three rounds of plaque purifica-tion. Genomic DNA of purified recombinant virus was extracted and electroporated into Escherichia coli DH10B. The ob-tained clone was confirmed by restriction enzyme digestion, PCR analysis and sequencing. DNA of positive clone was trans-virus with respect to virus titers. It was concluded that a complete genome of DEV( atten) was successfully cloned as an in-fectious bacterial artificial chromosome ( BAC) clone.