Study on the structure and function of viral proteins using artificial particles of human rotavirus

1. 利用杆状病毒表达系统制备了自组装的人工空单壳粒子（VP2/6），并对其进行了大量纯化。VP2/6颗粒伴粘膜佐剂（mLT-135、mLT-H44A、重组B亚基）经鼻免疫小鼠。mLT-135作为黏膜佐剂时，血清和粪便中IgG和IgA反应显著。免疫小鼠接种小鼠EW毒株后，mLT-135粪便中病毒抗原明显降低。此外，还观察到强烈的CTL反应。免疫小鼠脾细胞中检测到Th1或Th2细胞因子mRNA的表达。因此，人工空单壳颗粒与mLT-135联合有效地诱导了小鼠的体液和细胞免疫反应，表明人工颗粒和粘膜佐剂可作为人类抗轮状病毒感染的疫苗。我们观察到除G3血清型外，G血清型人轮状病毒也能引起哺乳小鼠腹泻。这一发现对人类轮状病毒毒株在小鼠体内的感染实验是有用的。为了建立轮状病毒的反向遗传学，我们进行了以下实验。(1)利用对胰蛋白酶依赖性较低的菌株SAT11-L2的VP4或VP7基因转录物，在高度依赖胰蛋白酶的人菌株KU的辅助病毒的帮助下，转染到MA 104细胞中。(2)制备了T7启动子-轮状病毒VP4或VP7基因- D型肝炎病毒的溶酶-T7终止子。将非复制必需的截断的NSP1基因与小核糖核酸病毒IRES和GFP基因连接，并插入到上述质粒中。将RNA转录物转染人KU菌株感染的MA 104细胞。建立了检测表达GFP或产生传染性重组病毒的细胞的筛选试验。然而，传染性人工轮状病毒迄今尚未被成功分离。少

1. Self-assembled, artificial and empty single-shelled particles (VP2/6) were prepared by using baculovirus expression system, and they were purified in a large quantity. VP2/6 particles accompanied with mucosal adjuvant (mLT-135, mLT-H44A, recombinant B subunit) were intranasally immunized in mice. Significant IgG and IgA responses in serum and stool were observed when mLT-135 was used as a mucosal adjuvant. In the inoculation with viruent murine strain EW for the immunized mice, a marked decrease of virus antigen in stools was observed in the case of mLT-135. Furthermore, a strong CTL response was also observed. Expression of mRNA for Th1 or Th2 cytokines was detected in the spleen cells from the immunized mice. Thus, artificial empty single-shelled particles combined with mLT-135 induced efficiently both of humoral and cellular immune responses in mice, suggesting the utility of the artificial particles and mucosal adjuvant as a vaccine for humans against rotavirus infection.2. We o … More bserved that human rotaviruses with G serotype other than G3 also induced diarrhea in suckling mice. This finding is useful for infection experiments using human rotavirus strains in mice.3. We carried out the following experiments for trying to establish reverse genetics of rotavirus.(1) RNA transcripts from VP4 or VP7 gene of strain SAT11-L2 which is less dependent on trypsin were transfected to MA 104 cells with the aid of a helper virus, human strain KU which is highly dependent on trypsin.(2) We prepared several constructs for transfection : T7 promoter-rotavirus VP4 or VP7 gene-rybozyme from type D hepatitis virus-T7 terminater. Truncated NSP1 genes which are not necessarily required for replication were connected to picornavirus IRES and GFP gene, and they were inserted in the above plasmid. The RNA transcripts were transfected into MA 104 cells infected with human strain KU.The screening assay for detecting the cells expressing GFP or producing infectious reassortant virus was developed. However, infectious artificial rotavirus has not been successfuly isolated so far. Less