人轮状病毒NSP4蛋白131和133位氨基酸变异对毒力影响的研究

目的研究两株A组人轮状病毒NSP4蛋白131位和133位氨基酸位点的变异对毒力的影响。方法从昆明地区2002年和2005年秋冬季婴幼儿腹泻流行期不同程度腹泻患儿中分离得到两株轮状病毒流行株02k38和05k44,RT-PCR扩增NSP4全长基因,进行cDNA序列测序。通过pGEX-5X-1载体,转化E.coliBL21以谷胱苷肽S-转移酶融合蛋白的形式表达两株病毒的NSP4蛋白C-端86～170位氨基酸（GST-NSP486-170）;并酶切融合头纯化得到两种NSP486-170蛋白,在ICR乳鼠中比较这两种蛋白致小鼠腹泻的差异。结果两种NSP486-170蛋白毒性无差异。结论两株轮状病毒131位和133位氨基酸位点的变异不影响毒力的改变,其致腹泻活性没有明显差异（P〉0.05）。

Expression of human rotavirus nonstructural proteins NSP4 and their different virulence in neonatal mice

Objective:To clarify the effect of genetic variation of NSP4 protein of two human rotavirus strains on their virulence.Methods:Two epidemic strains 02k38 and 05k44 were isolated from infants or babies with infection of human rotavirus in epidemic session in Kunming district in 2002 and 2005. The full-length NSP4 genes were amplified by RT-PCR and cDNA was sequenced. NSP486-170 amino acid at the C terminal fused with GST was expressed in E.coli BL21, and then the recombinant protein was purified with Glutathione SepharoseTM 4B affinity chromatography. GST was cut to obtain protein NSP486-170. Purified NSP4 protein was administered to ICR neonatal mice by intrapertioneal route and compared virulence.Results:The diarrhea inductive activity of NSP486-170 proteins that different at 131 and 133 residues were equally virulent.Conclusion:The mutation of 131 and 133 residues is not associated with altered virus virulence. We speculate that pivotal amino acid sequences in different NSP4 genotype are relative conservative.