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  

Construction of Transfer Vector of Marek’s Disease Virus Serotype 1 (MDV1) Containing F Factor of Eschericha coli and its Primary Application

作　　者：邱亚峰[1] 葛菲菲[1] 陈溥言[1] 

机构地区：[1]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

出　　处：《中国农业科学》2006年第11期2341-2346,共6页Scientia Agricultura Sinica

基　　金：国家863高新技术发展项目(2002AA245051)

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SphⅠ和NheⅠ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NheⅠ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。[ 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 was correct, and the sequences of the homologous recombinant left arm and right arm were correct. The two arms were connected with the reversed orientation. The positive plasmid, in which two loxP sites had the same orientation, was identified by Sph I and Nhe I ,named pUS-BGS. This linearizing vector by Nhe I was transfected into CEF, which was infected by CVI988 virus, by use of lipofectamine 2000. After three-cycle selection by MX-HAT, the infected CEF was stained with X-gal, blue plagues were appeared. [ Conclusion ] This research indicated that the transfer vector containing the E.coli F factor and two loxP sites, which had the same orientation, was constructed. And the results showed that this vector was efficient for the selection of recombinant MDV1. So this gave the basis for construction of the bacterial artificial chromosome of MDV 1.

关 键 词：马立克氏病病毒 细菌人工染色体 F因子 

分 类 号：S852.65[农业科学—基础兽医学]