应用Cre-loxP系统构建伪狂犬病病毒gE/TK双缺失株

Construction of a gE and TK Double Genes Deletion Mutant of Pseudorabies Virus by Cre-loxP System

为了构建无报告基因的猪伪狂犬病病毒(PRV)gE/TK双缺失株,以PRV-JSZ株为亲本毒株,增强型绿色荧光蛋白(EGFP)为报告基因,先构建出带有loxP位点的rPRV-gE-/GFP,通过Cre重组酶处理后,获得剔除EG-FP的gE单缺失株rPRV-gE-;再以rPRV-gE-为母本,以相同方法构建gE/TK双缺失株rPRV-gE-/TK-。荧光检测和PCR结果显示成功构建了gE单缺失株和gE/TK双缺失株;病毒生长曲线测定可见rPRV-gE-在PK15细胞上的增殖速度与野生株相似,而rPRV-gE-/TK-较为缓慢;rPRV-gE-/TK-对小鼠的半数致死量大于1×105 TCID50,显著高于rPRV-gE-和野生株;缺失株免疫小鼠强毒攻击后能提供80%的保护。这些结果表明以Cre/loxP系统可重复使用构建伪狂犬病病毒多基因缺失株,为研制伪狂犬病病毒基因缺失苗和载体疫苗提供了快速筛选的技术平台。

In order to construct a gE and TK double genes deletion mutant without a reporter gene, the recombinant virus rPRV-gE-/GFP with loxP sites was first constructed using PRV JSZ as a parental strain and an enhanced green fluorescent protein （EGFP） gene as a reporter gene. The recombinant virus rPRV-gE- was obtained by treating the genomic DNA of rPRV-gE-/GFP with Cre recombinase and transfecting PK15 cells. Then, the recombinant virus rPRV-gE-/TK- was constructed using rPRV-gE- as a parental strain. The results of fluorescence detection and PCR amplification showed that a gE gene deletion mutant and a gE/TK double genes deletion mutant were constructed successfully. The characteristics of the viruses revealed that rPRV-gE- had a similar growth curve in PK15 cells when compared with its wild type strain, but rPRV-gE-/TK- had a relatively slower growth speed. The mouse LD50 of rPRV-gE-/TK- was more than 1×105 TCID50, which was significantly higher than that of rPRV-gE- and wild type strain. The mice immunized with the rPRV-gE-/TK- provided an 80% protection against wild type virus challenge. These results indicated that a multiple gene deletion vaccine against PRV can be constructed by repeat use of Cre/loxP system, and it will provide a platform for the development of the recombinant attenuated PRV vaccine and its vectored vaccine.