中国安徽庐江鸭乙型肝炎病毒全基因组克隆酶谱分析和部分病毒基因正负单链探针的构建  

CLONING AND RESTRICTION MAPPING OF A CHINESE DUCK HEPATITIS B VIRUS GENOME AND SUBCLONING OF VIRAL SPECIFIC OPEN READING FRAMES INTO M13mp18 AND M13mp19

作　　者：杨文刚[1] 陈鸿珊[1] 

机构地区：[1]中国医学科学院医药生物技术研究所

出　　处：《病毒学报》1991年第3期210-216,共7页Chinese Journal of Virology

基　　金："七五"课题

摘　　要：用鸭乙型肝炎病毒(DHBV)阳性的安徽庐江鸭血清感染DHBV阴性的北京雏鸭,扩增病毒,将提取的DHBV-DNA插入pUC18质粒,转化E.coli JM 105。酶切重组质粒及South-ern转膜杂交结果证实,质粒pLJ76的插入片段为DHBV全基因组。用EcoR Ⅰ等11种限制性内切酶对pLJ76进行酶谱分析,并与美国,西德的已知DHBV基因组比较。定向克隆该株病毒不同基因编码区片段,构建正负单链探针,将斑点杂交和单链电泳检出的M13阳性重组子与已知序列的DHBV基因组作比较,提示获得了该株病毒基因组的S、Pre-S、P和X/C等蛋白编码区的正、负单链克隆株。Duck hepatitis B virus ( DHBV)in a single DHBV positive serum-number 76 from Lujiang county, China, was replicated in DHBV-DNA negative one day old Beijing ducklings. Viral DNA was purified, digested with EcoR I, ligated to pUC 18, transformed into E. coli JM 105. Using Southern transfer hybridization, we have obtained a clone, pLJ 76, containing 3.0 kb DHBV genome. Restriction mapping of this clone was constructed with 11 restriction enzymes. Compared with DHBV clones of USA and Germany, we found there were different digestion sites (Acc I, BamH I, EcoR V and Hind I I ) on the Chinese DHBV genome. Using low-melting point agarose DNA extraction method, we purified specific pLJ 76 DHBV-DNA fragments and subcloned them into Ml3-JMl05 system. Plus and minus strand probes of certain DHBV open reading frame including Pre-S, S, X/C and P were obtained. All of these probes were confirmed by DNA sequencing results.

关 键 词：DHBV 基因克隆 酶谱分析 单链 探针 

分 类 号：S852.65[农业科学—基础兽医学]