鸭乙型肝炎病毒全基因重组质粒构建及表达

目的构建鸭乙型肝炎病毒（DHBV）感染性克隆,通过体外细胞转染获得单一来源、基因序列清楚的体内实验的感染毒种。方法以湖北麻鸭DHBV分离株（DQ276978）为模板,用PCR法从该DHBV全基因组克隆质粒（pMD-DHBV）和DHBV阳性血清中获取3部分基因片段,总长4.4kb,克隆至载体PCR2.1的SacI和NotI酶切位点,构建含1.5倍鸭乙型肝炎病毒全基因的重组质粒,并酶切鉴定其插入方向。将构建的重组质粒经脂质体Lipo-fectineTM 2000导入鸡肝癌细胞系（LMH）细胞中转染表达,收集纯化后的转染细胞培养上清液作为体内实验感染的标准毒种。结果PCR鉴定、酶切鉴定及序列测定,证实成功获得正向插入的1.5倍鸭乙型肝炎病毒全基因重组质粒PCR2.1-DHBV1.5。Southernblot检测表明,该重组质粒在LMH细胞内存在各种病毒复制中间体;通过电镜观察,转染细胞上清液中存在DHBV完整病毒颗粒。结论经体外重组,构建出携带1.5倍加长DHBV基因组片段的重组质粒PCR2.1-DHBV1.5,并可在鸡肝癌细胞LMH中介导高水平病毒复制,从而获得含可自我复制病毒颗粒的转染细胞上清液作为动物体内感染接种物。

Construction of recombinant plasmid of duck hepatitis B virus and expression

Objective To construct the recombinant plasmid containing 1.5 - fold - overlength genome of DHBV and obtain the DHBV inocula collected from culture supernatant of DHBV transfected the avain LMH line. Methods 4.4 kb fragment of DHBV genome, derived from pMD - DHBV and DHBV positive serum, was cloned into Sac I and Not I site of the vector PCR2.1 to construct the recombinant plasmid PRC 2.1 - DHBV 1.5. And to identify its inserted direction by en- donucleases digestion. The avain LMH hepatoma cells were tmnsfected with plasmid PCR 2.1 - DHBV 1.5 by using LipofectineTM 2000 tmnsfection reagent. Results It was successful in the recombinant plasmid PRC 2.1 - DHBV 1.5 contain- ing 1.5 - fold - overlength genome of DHBV. All expected DHBV replicative intermediates were verified by Southern blot analysis in the supematant of LMH cells. And the DHBV could be observed by electron microscope. Conclusion The recom- binant plasmid PRC2.1 - DHBV1.5 containing a competent - replication, which initiates viral replication efficiently in infected avian hepatoma cells, is expected as a useful source of DHBV inoculum in vivo.