伪狂犬病病毒上海株（PRV—SH）gE^—／gI^—／GFP^＋缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus，PRV）上海株gI基因和gE基因克隆鉴定的基础上，采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstPI缺失掉gE基因5‘端363bp，同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分，并在下游 引入一个多克隆位点，构建了缺失转移载体pgEI-GFP。用DOTAP转染试剂盒将pgEI-GFP转染了感染PRV-SH的BHK-21细胞，待出现80％病变后收获病毒，并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。

Construction of Pseudorabies Virus SH Strain with gE-gI Gene Partial Deletion Mutant Including GFP Reporter Gene

On the basis of cloning and indentifying gE-gI gene of pseudurabie virus SH strain,the transfer plasmid vector was constucted in order to get the gE-gI gene partial deletion mutant.At first,gE gene and gI gene were cloned into pUC18,constructed the pgEI vector.Then, the 5'trminal sequence of gE gene was deleted 363bp using the restrict endonuclease in gE gene,The GFP expressing cassette was inserted into the deleting site. The recombinant plasmid pgEI including GFP reporter gene deleted part of gE-gI gene was constructed.BHK-21 cell which was infected with PRV-SH for 1-2h were tansfected with the complex of pgEI-GFP and DOTAPA deletion mutant was selected and purified many times in BHK-21 cell through GFP.Inoculation of mice with 2.0X107 PFU of the recombinant virus revealed that mice were partly protected against challenge with PRV-SH containing 2MLD.