含E.coli F因子的马立克氏病病毒1型（MDV1）转移载体的构建及初步应用.pdfVIP

中国农业科学 2006,39(11):2341-2346 Scientia Agricultura Sinica 含 E.coli F因子的马立克氏病病毒1型（MDV1）转移 载体的构建及初步应用 邱亚峰，葛菲菲，陈溥言 （南京农业大学动物医学院动物疫病诊断与免疫重点实验室，南京 210095 ） 摘要：【目的】为了构建 MDV1 细菌人工染色体，以便快速有效地进行重组病毒的筛选，进行了含E.coli F 因子的马立克氏病病毒 1 型（MDV1）转移载体的构建。【方法】利用 DNA 重组技术，构建了含有E.coli F 因子的 马立克氏病病毒1型（MDV1）转移载体，利用脂质体，将线性化的转移载体转染入已感染CVI988 病毒的CEF，用 MX-HAT 药物筛选 3 代以后，用 X-gal 染色,挑取阳性克隆。【结果】经测序分析发现，人工体外合成的单一 loxP 位点，序列正确，另外，同源重组左臂和右臂序列正确，并且相对连接。利用 Sph Ⅰ和Nhe Ⅰ双酶切，鉴定出含有 同向LoxP位点的转移载体，将其命名为pUS-BGS。将该载体用Nhe Ⅰ线性化后，用脂质体转染已感染CVI988病毒 的CEF，用MX-HAT药物筛选3 代以后，X-gal 染色，可以观察到呈现蓝色的重组病毒。【结论】本研究报道了含有 E.coli F因子以及两同向的loxP位点的MDV1转移载体的构建，利用该载体可以有效地进行重组病毒的筛选，为 下一步MDV1细菌人工染色体的构建奠定了基础。 关键词：马立克氏病病毒；细菌人工染色体；F因子 Construction of Transfer Vector of Marek’s Disease Virus Serotype 1 (MDV1) Containing F Factor of Eschericha coli and its Primary Application QIU Ya-feng, GE Fei-fei, CHEN Pu-yan (Key Laboratory of Animal Disease Diagnostic and Immunology ,Ministry of Agriculture, College of Veterinomry Medicine, Nanjing Agricultural University, Nanjing 210095) Abstract: 【Objective 】The objective was to construct the bacterial artificial chomosome containing the MDV1 genome, and to further develop quickly and efficiently the recombinant virus of MDV1 by using BAC vector. 【Method 】 Using the DNA recombinant techniques, the transfer vector containing E.coli F factor was constructed. The linearizing vector was transfected into CEF, which was infected by CVI988 virus. After a three-cycle selection by MX-HAT, the infected CEF was stained with X-gal, and the positive clones were picked. 【Results 】The sequence analysis showed that the loxP site synthesized in vitro w