We have established a hybridoma producing monoclonal antibody (MAb) against a linear epitope of glycoprotein (G protein) of the RC-HL strain of rabies virus. This MAbl5-13 showed almost the same neutralizing activity to all of five rabies fixed strains, including RC-HL, and reacted to the denatured G protein in western blot analysis. To characterize and map this linear epitope, an antigenic variant NR15-13 was selected from RC-HL strain in the presence of neutralizing MAbl5-13. The variant reacted with MAbl5-13 in an immunofluorescent antibody test but was not neutralized by the antibody and the antibody did not bind to the variant G protein in a Western blot analysis. The variant NR15-13 had an amino acid substitution at position 251 of the G protein, where tryptophan of the parental RC-HL strain was replaced by arginine. Site-directed mutagenesis analysis using the expression system in simian COS7 cells revealed that a single amino acid substitution at 251-tryptophan by arginine on the G protein of the parental RC-HL strain abolished the antigenicity of the epitope for MAbl5-13 in western blot analysis, and the replacement of 251-arginine by tryptophan recovered the activity. These results strongly suggest that tryptophan at position 251 on the G protein is essential for construction of the linear epitope against MAbl5-13.

A gene encoding the major inner capsid protein, VP6, of avian rotavirus was inserted into the eukaryotic expression vector PAX-91 under the control of the SRa promoter and was expressed at a high level in simian COS7 cells. The expressed VP6 was indistinguishable in terms of electrophoretic mobility from the corresponding protein synthesized in simian MA104 cells infected with avian rotavirus. Bindin,g assays with a series of monoclonal antibodies (mAbs) that corresponded to four antigenic sites on VP6 of avian rotavirus showed that the antigenic characteristics of the expressed product were identical to those of the native VP6 of avian rotavirus virions. Fiber-like structures that reacted strongly with antiserum against rotavirus were observed in VP6-expressing COS7 cells. Furthermore, an analysis of the tertiary structure of the expressed VP6 protein indicated that it adopts a trimeric configuration, similar to that of the major inner capsid protein of PO-13 virus. From these tisults, it appears that recombinant VP6 will facilitate studies of the structure and function of authentic VP6, an important protein in avian rotavirus.

The cDNA encoding the VP6 gene of avian rotavirus PO-13 strain was inserted into the bacterial expression vector pET-3a. Upon isopropyl-1-thio-13-D-galactoside induction, the E. coti BL21 (DE3) harboring the vector containing cDNA of the VP6 gene produced an approximately 45-kDa polypeptide, which reacted with rabbit serum against PO-13 strain in Western blotting. To study the antigenic sites on VP6, various deletion mutants were constructed, expressed in E. coli and the reactivity with antigenic site I- and Iispecific MAbs analyzed by Western blotting. Site I, which is shared with all group A mammalian and avian rotaviruses except for chicken rotavirus, was found to be located at amino acid positions 45 to 65, and site II, which probably contributes to an authentic group A antigen common to both mammalian and avian rotaviruses, at amino acid positions 134 to 142.

本试验利用杆状病毒载体成功地表达狂犬病病毒糖（G）蛋白。从转染重组了日本动物用狂犬病病毒疫苗株RCHL株G蛋白基因的转移载体和野生型杆状病毒DNA的Sf-9细胞中，分离出2个重组杆状病毒克隆。应用印渍法和荧光抗体法（IFA）从重组杆状病毒感染的Sf-9细胞检出了狂犬病毒G蛋白。Sf-9细胞表达的G蛋白与狂犬病毒RCHL株感染NA细胞的G蛋白的分子量一致。35个抗RCHL株G蛋白的单克隆抗体均与重组杆状病毒感染的Sf-9细胞反应，表明表达的G蛋白的抗原性没有发生变异。表达的G蛋白主要分布在Sf-9细胞的膜表面，形成类似环状的荧光，这种荧光与RCHL株感染的NA细胞的荧光相同。