CAJ | 학술논문

在已构建鸡贫血病毒（CAV）全基因组体外克隆（pCAV2.4）的基础上，设计一对特定引物，用PCR定向点突变方法扩增出含有完整的EcoRI位点的CAV全基因组（2.3kb），并克隆入pUC18载体中，获得阳性克隆，命名为pCAVE+。经酶切鉴定和序列分析EcoRI位点两侧序列，原先不完整的EcoRI位点（-GACTTC-）已被定向点突变为完整的EcoRI位点（-GAATTC-）。对重组质粒pCAVE+以EcoRI酶切回收CAV全基因组DNA，在体外连接并经纯化后，经Effectene转染易感宿主细胞MSB1。将转染细胞培养上清连续传代8次以后，出现病毒复制（MSB1细胞培养基颜色不再变黄）。取此时细胞培养液感染无CAV的1日龄SPF小鸡，14天出现典型的鸡贫血症状和胸腺萎缩、骨髓脂肪变性等病变。获得了能在体外自主复制并组装成完整CAV病毒子的感染性核酸。
재이구건계빈혈병독（CAV）전기인조체외극륭（pCAV2.4）적기출상，설계일대특정인물，용PCR정향점돌변방법확증출함유완정적EcoRI위점적CAV전기인조（2.3kb），병극륭입pUC18재체중，획득양성극륭，명명위pCAVE+。경매절감정화서렬분석EcoRI위점량측서렬，원선불완정적EcoRI위점（-GACTTC-）이피정향점돌변위완정적EcoRI위점（-GAATTC-）。대중조질립pCAVE+이EcoRI매절회수CAV전기인조DNA，재체외련접병경순화후，경Effectene전염역감숙주세포MSB1。장전염세포배양상청련속전대8차이후，출현병독복제（MSB1세포배양기안색불재변황）。취차시세포배양액감염무CAV적1일령SPF소계，14천출현전형적계빈혈증상화흉선위축、골수지방변성등병변。획득료능재체외자주복제병조장성완정CAV병독자적감염성핵산。
Using the recombinant plasmid （pCAV2.4） as template DNA，chickenanaemia virus （CAV） genomic DNA was amplified by polymerase chain reaction and cloned into pUC18 vector （pCAVE+）.The incomplete EcoRI recognized sequence （－GACTTA－） in pCAV2.4 was point-mutated into the complete EcoRI recognized sequence （－GAATTC－） in pCAVE＋.The 2.3kb CAV genomic DNA was extracted from the recombinant pCAVE+ at the sites of EcoRI，then re-ligated，and transfected into host cell line MSB1.After 8 passages，the cell culture medium didn’t turn yellow.The supernatant was inoculated into 1 day-old SPF chickens，typical anaemia and thymus atrophy were shown after 14 days.This study showed CAV genomic DNA can be transformed into infectious virus.