新城疫病毒F_（48）E_8株cDNA文库的构建

CONSTRUCTION OF cDNA LIBRARIES OF A CHINESE VIRULENT NEWCASTLE DISEASE VIRUS STRAIN F_(48)E_8

以ＳＰＦ鸡胚繁殖我国标准新城疫病毒Ｆ＿（４８）Ｅ＿８强毒株，经蔗糖密度梯度超速离心纯化病毒。再以酚－ＳＤＳ法提取病毒基因组ＲＮＡ，作为反应模板、采用Ｐｒｏｍｅｇａ公司商品试剂盒合成双股ｃＤＮＡ。以同聚物加尾的方法将ｃＤＮＡ克隆到质粒ｐＧＥＭ３Ｚｆ（一）中，经ＡＩＸ平板筛选，限制性内切酶分析和Ｄｉｇｏｘｉｇｅｎｉｎ标记的核酸探针检测，共获得插入外源片段大小在０．６～４．８ｋｂ的阳性克隆７５个。

The virulent strain F_(48)E_8 of Newcastle disease virus(NDV)was grown in SPF chicken eggs and purified bycentrifugation in a sucrose gradient,then extracted with phenol-SDS to gain genomic RNA that was used as template togenerate cDNA with RiboClone cDNA synthesis system of Promega Corporation. The two-strand cDNA was tailed with oli-go(dC)and annealed to plasmid pGEM 3Zf(一)which was cut with PstI and tailed with oligo(dG ). The recombinantswere transformed into comptent E. Coli., and 75 positive clones which inserted 0.6～4. 8kb cDNA fragments werescreened with AIX plates and dot-blot hybridization by Digoxigenin labeled probes.