两株Wa株人源轮状病毒NSP4蛋白的表达及其致ICR小鼠腹泻的毒力比较

目的研究两株Wa株人源轮状病毒NSP4蛋白139位氨基酸位点的变异对毒力的影响。方法从2002～2004年昆明地区婴幼儿轮状病毒感染者大便样品中,通过RT-PCR,PCR扩增了22株轮状病毒株的NSP4全长基因、cDNA序列,并对cDNA序列测序,发现这些病毒株的NSP4氨基酸序列高度保守,仅在79位、139位氨基酸位点存在差异。选取139位氨基酸不同(V/I)的两株病毒株,在E.coli BL21中,用pGEX5X-1载体融合表达了两株病毒的86～175位氨基酸(GST-NSP4_(86-175);并酶切融合头得到两种NSP4_(86-175)蛋白,在ICR乳鼠中比较这4种不同蛋白致小鼠腹泻的差异。结果发现GST-NSP4_(86-175)蛋白毒性比NSP4_(86-175)蛋白强,而139位氨基酸不同(V/I)的两株病毒株的NSP4_(86-175)蛋白毒性没有明显的差异。结论139位氨基酸位点的变异并不影响NSP4蛋白的毒力改变。

NSP4 Proteins Expression of Two Wa Strains of Human Rotavirus and Their Different Virulence in ICR Neonatal Mice

Objective To examine the effect of genetic variation of NSP4 protein of two human rotavirus strains on their virulence. Methods From 2002-2004, stool samples were collected from infants or babies with infection of human rotavirus in Kunming district. By using RT-PCR, the fulllength NSP4 genes and cDNA sequences of 22 strains of viruses were amplified and cDNA was sequenced. These NSP4 sequences were found to be closely related to each other and there were differences at the 79th, 139th amino acid residues. By using pGEX5X-1 vector, 86-175 amino acid residues of 2 strains were expressed. The sequences of the NSP4 were different at 139th residue in E. coli BL-21. GST fusion proteins GST-NSP4_(86-175) were purified. GST was cut to obtain protein NSP4_(86-175). To compare their virulence, these 4 purified proteins were used to induce diarrhea in ICR neonatal mice by intraperitoneal administration. Results GST-NSP4_(86-175) proteins were more virulent than NSP4_(86-175) proteins and two NSP4_(86-175) proteins that were different at the 139th residue were equally virulent. Conclusion The mutation of 139th residue is not associated with altered virus virulence.