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作者用双体根瘤土壤杆菌载体RG-2构建了RGRgp,其中包括花椰菜花叶病毒35s启动子控制下的狂犬病病毒ERA株完整的未经修饰的糖(G)蛋白基因,以及能在卡那霉素培养基中选择生长的新霉素磷酸转移酶Ⅱ。将RGRgp通过土壤杆菌介导转化番茄植物子叶,并在选择性培养基中作组织培养。经聚合酶链反应(PCR)证实,得到4株含数个RGRgp构建物拷贝的转基因番茄。RNA印迹法分析表明其中两株的叶和果实表达G蛋白RNA。用两种兔抗狂犬病病毒G蛋白的多克隆抗血清免疫沉淀纯化叶和果实组织抽提物中的重组G蛋白,出现两条蛋白沉淀带,相应分子量为62.5和60.0kDa。在BHK细胞中培养的变性狂犬病病毒G蛋白出现
The authors constructed the RGRgp with the Agrobacterium tumefaciens vector RG-2, which contains the complete unmodified sugar (G) protein gene of the rabies virus ERA strain under the control of the cauliflower mosaic virus 35s promoter, Neomycin phosphotransferase II, a growth medium of choice for growth medium. The RGRgp was transformed into tomato plant cotyledons by Agrobacterium tumefaciens and cultured in selective medium for tissue culture. Confirmed by polymerase chain reaction (PCR), four transgenic tomato plants containing several copies of the RGRgp construct were obtained. Northern blot analysis showed that two of the leaves and fruits expressed G protein RNA. Purification of recombinant G protein from extracts of leaf and fruit tissue using two polyclonal antisera to rabbit anti-rabies virus G protein revealed two protein precipitation bands with corresponding molecular weights of 62.5 and 60.0 kDa. The degenerative rabies virus G protein, which is cultured in BHK cells, appears