高致病性猪繁殖与呼吸综合征病毒NSP1蛋白重组腺病毒的构建及其对小鼠的免疫抑制作用

Immunosuppression of NSP1 of highly pathogenic porcine reproductive and respiratory syndrome virus expressed in a recombinant adenovirus in mice

利用RT-PCR扩增获得高致病性猪繁殖与呼吸综合征病毒(PRRSV)SY0608株非结构蛋白基因NSP1,将其克隆至穿梭载体pShuttle-CMV,经PmeⅠ线性化后在BJ5183大肠杆菌内与腺病毒骨架载体pAdEasy-1同源重组,获得重组腺病毒载体,再经PacⅠ线性化后转染至HEK-293A细胞,获得重组腺病毒,IFA和Western-blot鉴定结果证明其可以表达NSP1。将该重组腺病毒2次接种ICR小鼠,每次间隔2周,于免疫后不同时间取脾淋巴细胞进行体外培养,用于淋巴细胞增殖试验,并用ELISA检测IFN-γ和IL-10水平。结果显示,与野生型腺病毒(wtAd)或DMEM对照组相比,重组腺病毒rAd-NSP1组T淋巴细胞增殖指数(SI)和IFN-γ水平显著降低,IL-10水平明显增高(P<0.05)。结果表明,高致病性PRRSV NSP1蛋白具有明显的免疫抑制作用。

The non-structural protein I（NSP1） gene of the highly pathogenic PRRSV strain SY0608 was amplified by RT-PCR and cloned into pShuttle-CMV plasmid, and confirmed by PCR and sequence analysis. The recombinant plasmid was linearized and transformed into E. coli BJ5183 strain together with adenovirus backbone vector pAdEasy-1. After being linearized with Pine I , the recombinant adenovirus plasmid pAd-NSP1 was transferred into HEK293-A cells and the recombinant adenovirus rAd-NSP1 was obtained. The results of Western-blot and IFA showed that the NSP1 could be expressed in 293 cells line infected with the recombinant adenovirus. Forty-five ICR mice were inoculated with rAd-NSP1 subcutaneously twice at 2-week apart, and PRRSV-specific cell mediate immune responses were detected by T lymphocyte proliferation assay and ELISA for IFN-γ and IL-10. The results showed that the mice inoculated with rAd-NSP1 had significantly lower levels of lymphocyte proliferation and IFN-γ, but had obviously higher levels of IL-10 compared with the control group inoculated with wild type adenovirus or medium alone. It indicated that NSP1 of highly pathogenic PRRSV could inhibit the immunity in mice.