猪轮状病毒NSP4蛋白135、136和138位点氨基酸突变对毒力影响的研究

Expression of porcine rotavirus nonstructural proteins NSP4 and their different pathogenic effect in neonatal mice

在比较我国猪轮状病毒(PRV)强弱毒株非结构蛋白NSP4 cDNA序列时发现,两者在可能与致病性有关的区域(aa112～aa175)内存在显著差异。为进一步研究这种变异是否与毒力改变有关,本研究从我国流行弱毒株JL94中扩增Nsp4基因并突变其135、136和138氨基酸位点,以谷胱甘肽S-转移酶GST重组蛋白的形式在大肠杆菌Rosseta中表达突变和未突变两种Nsp4蛋白C端编码aa 112～aa 175的截短蛋白并用Glutathione SepharoseTM4B亲和纯化。将纯化的两种蛋白均以40μg的剂量腹腔接种新生BALB/c乳鼠。结果显示,接种GST-Nsp4突变重组蛋白组,有大部分小鼠先后发生腹泻(7/12),接种未突变GST-Nsp4重组蛋白组,只有少部分小鼠发生先后腹泻(3/12),而注射PBS的对照组,均未发生腹泻。本研究证明PRV Nsp4第135位、136位和138位氨基酸位点的变异影响蛋白毒性的改变,其致腹泻活性具有明显差异(p<0.05)。为进一步研究PRV的致腹泻机理和防制及其疫苗的研制奠定基础。

Porcine rotavirus （PRV） is one of the agents that causes diarrhea in piglets and the pathogenic PRV was associated with the nonstructural protein of NSP4. To further investigate the function of NSP4 in the pathogenesis of PRV, the fragments of NSP4 gene from a low pathogenic PRV strain JL94 was cloned （non-mutated） or synthesized with encoding sequence mutations at aa135, aa136 and aa138, and both mutated and non-mutated truncated NSP4 were expressed in E.coli and purified with Glutathione SepharoseTM 4B affinity chromatograph. Furthermore, 6 day old BALB/c mice were inoculated intraperitoneally with the mutated or non-mutated truncated NSP4 （40 Ixg for each mouse）, respectively. The results showed that non-mutated NSP4 only induced diarrhea in 3 of 12 mice, while diarrhea occurred in 7 of 12 mice inoculated with mutated NSP4. In conclusion, the pathogenic effect of PRV was closely related to the amino acids of aa135, aa136 and aa138 in the NSP4.