利用CRISPR-Cas9技术和Cre/loxP系统构建猪伪狂犬病病毒gE基因缺失疫苗株

Construction of a gE gene-deleted vaccine strain of porcine pseudorabies virus by the CRISPR-Cas9 technology and the Cre/loxP system

通过对4株伪狂犬病病毒(PRV)流行株进行比较,选择免疫原性最高的HNQYY2012为亲本株,利用CRISPR-Cas9技术和Cre/loxP系统构建带有LoxP位点的HNQYY2012ΔgE/EGFP^+,然后利用环化重组酶(Cre)去除增强型绿色荧光蛋白(EGFP)基因,构建PRV gE基因缺失株HNQYY2012ΔgE。PCR鉴定和测序结果表明,PRV gE基因缺失株构建成功;一步生长曲线表明,HNQYY2012ΔgE增殖速度慢于亲本株,但最高滴度与亲本株相近;HNQYY2012ΔgE和亲本株对小鼠的半数致死量分别为10^3.8TCID50和10^2.5TCID50,表明gE基因缺失株毒力降低。制备的灭活疫苗免疫小鼠能完全抵御5LD50和10LD50强毒攻毒。本文利用CRISPR-Cas9技术和Cre/loxP系统成功构建PRV gE基因缺失株HNQYY2012ΔgE,为猪伪狂犬病灭活疫苗的研制提供了参考。

By comparing 4 isolates of porcine pseudorabies virus(PRV),the isolate HNQYY2012 with the highest immunogenicity was selected as the parental strain to construct HNQYY2012ΔgE/EGFP+with a loxP site.Utilizing the CRISPR-Cas9 technology and the Cre/loxP system,and then removing the EGFP by Cre recombinase,the gE gene-deleted strain HNQYY2012ΔgE of porcine pseudorabies virus was constructed.The PCR identification and sequencing results indicated that the gE gene-deleted strain HNQYY2012ΔgE of porcine pseudorabies virus was successfully constructed.The results of the one-step growth curves showed that the growth rate of HNQYY2012ΔgE was slower than that of the parental strain,and the highest virus titer was similar to that of the parental strain.The median lethal doses of HNQYY2012ΔgE and the parental strain to mice were 10^3.8 TCID50 and 10^2.5 TCID50,respectively,which indicated that the virulence of the gE gene-deleted strain decreased.The KM mice immunized with the inactivated vaccine completely resisted against the attack of 5 LD50 and 10 LD50.In summary,the gE gene-deleted strain HNQYY2012ΔgE of porcine pseudorabies virus was successfully constructed by the CRISPR-Cas9 technology and the Cre/loxP system,which provided a foundation for the development of an inactivated vaccine against porcine pseudorabies virus.