【摘要】 本试验用甘油-酒石酸钾密度梯度离心纯化了兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV),所得病毒制剂实心颗粒占90％,没有宿主核酸污染。在常规SDS-蛋白酶K法的基础上,建立了一种耗时短、重复性高的RHDV核酸抽提程序。按照细小病毒基因组的特性,在体外合成了RHDV dsDNA,证明兔出血症病毒基因组是具有3′端发夹结构的ssDNA。以核酸酶S1修饰RHDV dsDNA,将其克隆入BS质粒载体,转化大肠杆菌DH5株。经筛选鉴定,共得RHDV DNA克隆23个,其中最大者(pYC-2)为4.6kb。选用18种限制性内切酶作酶谱分析,结果表明,12种酶在pYC-2克隆基因上不存在切点;以双酶法,用Cla Ⅰ、HincⅡ,Pvu Ⅰ、Bgl Ⅱ和Hpa Ⅰ绘制出pYC-2DNA的酶切图谱。更多还原

【Abstract】 The RHDV virions were purified by means of chloroform treatment, PEG precipitation, differential centrifugation and Potassium Tartrate Glycerol density gradient centrif ugation. The RHDV ssDNA was extracted from the purified viral preparation with SDS-Proteinase K, phenol and chloroform, RHDV dsDNA was then synthesized in vitro by E.coli DNA polymerase I Klenow large fragment from RHDV ssDNA with its 3’ hairpin as template-primer. Having been modified by nuclease SI, RHDV dsDNA was ligated to Bluescript plasmid vector cleaved by EcoR V. After transformed by the ligation mixture, E.coli DH5 competent cells were cultured in LB medium, and the white colonies were screened on selective medium. Recombinant plasmids were extracted and subjected to the agarose electrophoresis identification. Consequently, 23 clones, 1-4.6kb in length, were selected. The physical map of pYC-2 was made by restricted enzymes Cla I , Hinc I , Pvu I , Bgl I and Hpa I . These results demonstrate that RHDV, which genome formerly characterized as dsDNA, is a ssDNA virus.更多还原