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制备香蕉线条病毒广东分离物(BSV-GD)的抗血清,可为BSV-GD的快速检测和进一步研究BSV编码蛋白的功能提供条件。通过常规分子生物学方法,克隆了BSV-GD衣壳蛋白(Coat protein,CP)功能域基因,构建了该基因的原核表达载体pET28b-CP,并诱导表达了大小约为46.5 kD的融合蛋白His-CP。可溶性分析表明该融合蛋白以包涵体形式存在。利用His标签纯化试剂盒对目的蛋白进行纯化、回收,获得了高纯度的融合蛋白。以纯化的融合蛋白为抗原免疫健康大耳白兔,成功制备了BSV-GD CP功能域基因编码蛋白的兔抗血清。Western-blotting分析表明该抗血清具有很强的特异性,间接ELISA法检测抗血清效价达204 800倍以上,对植物材料的合适检测浓度为1︰1 600~1︰6 400。
Preparation of antiserum against banana streak virus Guangdong isolate (BSV-GD) provides conditions for the rapid detection of BSV-GD and further study of the function of BSV encoded proteins. The gene of BSV-GD capsid protein (CP) was cloned by conventional molecular biology method. The prokaryotic expression vector pET28b-CP was constructed and the fusion protein His was induced to a size of 46.5 kD -CP. Soluble analysis showed that the fusion protein was in the form of inclusion bodies. The target protein was purified and recovered by using His-tag purification kit, and a high-purity fusion protein was obtained. Rabbit antisera were prepared by immunizing rabbits with purified fusion protein as antigen. Western-blotting analysis showed that the antiserum was highly specific. The titer of the antiserum was detected more than 204 800 times by indirect ELISA, and the suitable detection concentration for plant material was 1︰1 600 ~ 1︰6 400.