Development of the system for the formation of infectious particles of rotavirus with the aid of genetic engineering

在研究项目的最后阶段，我们成功地开发了轮状病毒的反向遗传系统，尽管我们必须为该系统使用辅助病毒。开发方法如下。1)我们制备了一个质粒pT 7/VP 4-SA 11-L2，其中插入了来自胰蛋白酶非依赖性猴轮状病毒SA 11-L2克隆的VP 4基因，该克隆具有在培养细胞中有效增殖的能力。2)将表达T7 RNA聚合酶的牛痘病毒感染到COST细胞中。3)转染质粒pT 7/VP 4（SA 11-L2）。4)将作为辅助病毒的人轮状病毒株KU感染到COST细胞中，并将细胞培养两天。5)将来自病毒感染细胞的培养液感染MA-104细胞，并在特异性抑制辅助病毒KU生长的中和性单克隆抗体YO-2C 2存在下维持细胞2天。6)从培养液中提取的RNA经聚丙烯酰胺凝胶电泳证实了RNA的电泳图谱，从而成功地制备了人工插入VP 4基因的感染性轮状病毒颗粒。接下来，我们制备了具有人工突变的VP 4基因的感染性轮状病毒。本研究通过对VP 4基因进行突变，去除了VP 4基因上的Pst Ⅰ酶切位点（pT 7/VP 4（SA 11-L2）-Δ Pst Ⅰ），去除了VP 4基因上的Hae Ⅱ酶切位点（pT 7/VP 4（SA 11-L2）-Δ Hae Ⅱ），从而首次建立了轮状病毒反向遗传学系统。我们尝试用突变的VP 7基因或NSP 1基因制备感染性轮状病毒，并在不久的将来用质粒DNA制备感染性轮状病毒。

At the final stage of the research project, we succeeded in the development of the system of reverse genetics of rotavirus, although we have to employ a helper virus for the system. The methodology for the development is the following. 1) We prepared a plasmid (termed as pT7/VP4-SA11-L2) which the VP4 gene from a trypsin-independent simian rotavirus SA11-L2 clone with the ability of efficient multiplicity in cultured cell was inserted. 2) Vaccinia virus expressing T7 RNA polymerase was infected into COST cells. 3) A plasmid pT7/VP4(SA11-L2) was transfected. 4) A human rotavirus strain KU as a helper virus was infected into COST cells, and the cells were cultured for two days. 5) The culture fluid from the virus-infected cells was infected to MA-104 cells, and the cells were maintained for two days in the presence of a neutralizing monoclonal antibody YO-2C2, which inhibits specifically the growth of a helper virus KU. 6) RNA pattern of the RNA extracted from the culture fluid was confirmed by polyacrylamide gel electrophoresis.By these procedures, we succeeded in the preparation of infectious rotavirus particles which the VP4 gene was artificially inserted. Next, we prepared infectious rotaviruses with artificially mutated VP4 gene. The mutated VP4 genes are the one (pT7/VP4(SA11-L2)-Δ PstI) which a PstI restriction site on the gene was removed, and the other (pT7/VP4(SA11-L2)-Δ HaeII) whose a HaeII restriction site was removed by mutagenesis.Thus, we first developed the reverse genetics system for rotavirus. We try to prepare the infectious rotavirus with the mutated VP7 gene or NSP1 gene, and to prepare the infectious rotavirus with the aid of only plasmid DNAs in near future.