应用Cre/loxp系统构建含有BAC序列的重组伪狂犬病毒

摘要

近年来我国出现伪狂犬病毒变异株,导致猪伪狂犬病重新爆发流行.为研究伪狂犬病毒变异株毒力增强与抗原变异的分子机制,需要建立该病毒的感染性克隆操作系统.本研究通过构建转移载体pTEGGF,利用同源重组将含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)表达框及两翼各一个loxp位点,插入到伪狂犬病毒变异株PRV JS-2012 gG编码区下游,获得重组病毒rJS2012-gG/EGFP.将表达Cre重组酶的质粒pcDNA3.1-Cre转染BHK-21细胞,再感染rJS2012-gG/EGFP,筛选获得含有单一loxp位点的重组病毒rJS2012-gG/loxp.将rJS2012-gG/loxp基因组、含有EGFP标记基因的BAC载体pBeloBAC11-EGFP和pcDNA3.1-cre共转染BHK-21,获得含有BAC序列插入的重组病毒rJS2012-BAC.一步生长曲线与空斑试验显示,重组病毒rJS2012-BAC在体外生长略慢于亲本病毒.本研究成功构建了含有BAC载体的重组伪狂犬病毒,为进一步建立伪狂犬病毒变异株的感染性克隆操作系统,开展变异株的分子病原学研究打下良好基础.%To develop a reverse genetic system for Pseudorabies virus (PRV), a recombinant PRV carrying a BAC sequence was constructed in the present study. An EGFP expression cassette flanked by a loxp site in both sides was inserted into the downstream of gG coding region of a pseudorabies virus variant strain PRV JS-2012 by homologous recombination, generating a recombinant virus rJS2012-gG/EGFP. BHK-21 cells were transfected with the pcDNA3.1-Cre that expressed Cre recombinase and then infected with rJS2012-gG/EGFP. A recombinant virus rJS2012- gG/loxp carrying only one loxp site downstream of gG coding region was obtained. Subsequently, the genome of rJS2012-gG/loxp was co-transfected with pcDNA3.1-cre and pBeloBAC11-EGFP into BHK-21 cells, generating a recombinant virus rJS2012-BAC. The recombinant virus rJS2012-BAC carried the BAC vector sequences in its genome and grew slightly slower than its parent strain JS-2012 in vitro. The availability of the recombinant virus rJS2012-BAC would contribute to developing a reverse genetic system for PRV.