香蕉线条病毒衣壳蛋白功能域基因的原核表达及抗血清制备

Prokaryotic Expression and Antiserum Preparation of Functional Domain of Banana streak virus Coat Protein Gene

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（Coatprotein，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46．5kD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，成功制各了BSV-GDcP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204800倍以上，对植物材料的合适检测浓度为1：1600～1：6400。

In order to provide rapid detection of Banana streak virus Guangdong isolate （BSV-GD） and to study the virus＇ gene function, BSV-GD coat protein （CP） functional domain was cloned, and prokaryotic expression recombinant plasmid pET28b-CP was constructed by inserting the cloned gene into the plasmid pET-28b （＋） . Induced by IPTG, Escherichia coli Rosseta （DE3） containing pET28b-CP produced the fusion protein His-CP about 46.5 kD in size. Soluble analysis of the fusion protein indicated that it was in the inclusion body. The highly purified interest protein was obtained by using the His tag purification kit. The special polyclonal antibody was generated through the purified protein immunizing healthy big ear rabbits. Western-blotting analysis showed that the antiserum has very strong specificity. Indirect enzyme immunoassay suggested the antiserum titer was higher than 1 ： 204 800. The available antiserum concentration for virus detection from plant materials was 1 ： 1 600 - 1 ： 6 400.