032基因缺失猪痘病毒载体的构建及鉴定

Construction and Identification of the 032 Gene Deletion in Swinepox Virus

目的：构建毒力减弱的猪痘病毒载体。方法：以猪痘病毒野生毒株NT1501株为亲本毒株,通过同源重组方法,构建缺失032毒力基因的猪痘病毒SPV△032。检测SPV△032在不同种属来源细胞系中的培养滴度,并通过动物试验来检测其毒力。结果：猪痘病毒SPV△032株与野生NT1501株在PK15和ST等猪源细胞中的培养滴度差异不显著,均可达到5.0×106TCID50左右。猪痘病毒SPV△032株在Vero、MDBK等其他物种来源的细胞系中不能进行复制,连续传代2-3代以后,细胞培养物中检测不到猪痘病毒基因,与野生NT1501株培养结果一致。动物试验结果表明,用高浓度SPV△032接种实验猪,接种部位皮肤未出现任何症状,比野生NT1501株的毒力显著降低。接种SPV△032实验猪的细胞免疫和体液免疫反应水平均显著高于野生NT1501株（P〈0.05）。接种SPV△032的实验猪不会通过粪便等途径向环境中排毒。结论：本研究构建了032基因缺失猪痘病毒SPV△032,该病毒的毒力与野生NT1501株相比显著下降,可以作为弱毒猪痘病毒载体,用于兽用疫苗研发。

Objective：To construct an attenuated swinepox virus（SPV） vector. Methods：The 032 gene deletion SPV△032 was constructed by homologous recombination with SPV NT1501 wild strain. The titers of the SPV△032 culture in different species-derived cell lines were measured,and the virulence of the SPV△032 was detected by animal experiment. Results：There was no significant difference in the culture titers between the SPV△032 strain and the wild NT1501 strain in PK15 and ST cell lines,and the culture titer of the SPV△032 was about 5. 0 × 106TCID50. The SPV△032 could not be replicated in Vero,MDBK and other species-derived cell lines. After 2-3 passages,the swinepox virus gene could not be detected in the cell culture of the SPV△032,which was consistent with the culture of wild NT1501 strain. The results of animal experiments showed that the inoculation site of the SPV△032 inoculated pigs with high concentration did not show any symptoms,and the vi-rulence of the SPV△032 was significantly lower than that of the wild NT1501 strain. The cellular immune response and humoral immune response of the SPV △032 inoculated pigs were significantly higher than those of wild NT1501 strain inoculated pigs（P〈0. 05）. The SPV△032 virus will not give emission to the environment through the feces of the SPV△032 inoculated pigs. Conclusion：The 032 gene deletion SPV△032 was constructed in this study. The virulence of the SPV△032 was significantly lower than that of the wild NT1501 strain,andcould be used as attenuated vector in veterinary vaccine development.