鸡痘病毒基因组DNA复制非必需片段的鉴定

IDENTIFICAT1ON OF NONESSENTIAL REGIONS INGENOMIC DNA OF FOWLPOX VIRUSOF CHINESEVACCINE STRAIN

将中国鸡痘病毒鹌鹑化弱毒株（２８２Ｅ４）蚀斑纯化，在ＳＰＦ鸡胚成纤维细胞上增殖后，经超声波裂解，蔗糖梯度离心得到纯化病毒，以蛋白酶Ｋ消化，酚：氯仿、氯仿和水饱和乙醚抽捉，乙醇沉淀，得到ＦＰＶ基因组ＤＮＡ，后用ＥｃｏＲｌ消化，随机克隆到ｐＵＣ１８质粒中。限制性内切酶片段电泳分析结果表明，克隆到１０个不同大小的片段，根据酶切图谱分析，选取克隆或亚克隆后的单一酶切位点，插入标记基因（Ｐ１１一ＩａｃＤ筛选出３个复制非必需片段。为进一步构建表达外源片段重组质粒载体创造了良好的条件。

ln this paper we report the identification of three nonessential regions of fowlpox virus(FPV)genome of Chinese vaccine strain 282E4.The virus was plaque一purified and grown in chick embryo fibrob-last);Harvested virus preparation was sonated and purfied by sucrose density gradlent centrifugatlon。After proteinase K dlgestion,genomic DNA was extracted by phenol:chIoroform,chloroform and water一saturated ether,and precipitated by ethanol. FPV DNA EcoR I fragments were randomly cloned into pUCI8and l0 clones with inserted DNA of varylng size were obtained.Clones or subclones which had single cleavable site were inserted by the reported gene Pl l一lacZ;Then purified plasmid DNA was used to transfectCEF which had been infected by FPV。Recombinants with blue plaques were selected and purified 3 times inCEF cultures overlaid with agrose containing X一Gal. Three cloned FPV fragments were identified asnonessential regions for virus replication。