香蕉束顶病毒海口分离物Rb蛋白的原核表达、纯化及多克隆抗血清制备

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的E.coli BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结果表明,表达产物大小为22.6ku,且主要以包涵体形式稳定表达。目的蛋白经Ni2+-NTA琼脂糖亲和层析纯化后免疫家兔并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的BBTV编码Rb蛋白发生特异性反应。间接ELISA法测定的抗血清效价大于1:250000。用抗血清对感病香蕉和健康香蕉进行检测,结果表明,抗血清对感病香蕉有很高特异性,最佳工作浓度为1:1500。

Prokaryotic Expression and Purification of Rb Protein of Banana Bunchy Top Virus HaiKou Isolate and Preparation of Polyclonal Antiserum Against Rb

The opening reading frame of Rb gene of Banana bunchy top virus was cloned by polymerase chain reaction(PCR)using total DNA from pseudostem and leaves of banana infected with the virus as a template. The Rb gene was inserted into prokaryotic expression vector pET-28b to produce a recombinant plasmid pET28b-Rb. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) and expressed with IPTG induction. The result of SDS-PAGE showed that the fusion protein with a molecular weight approximately 22.6 ku expressed steadily as an inclusion body. The recombinant Rb protein was purified with Ni2+-NTA agarose affinity chromatography, and used to immune the rabbit for antiserum preparation. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the Rb of BBTV. The titer of the antiserum was up to 1/250 000 by indirected enzyme-linked immunosorbant assay(ID-ELISA). According to the result of the detection of ID-ELISA, the antiserum had a high specificity to leaves infected with BBTV under the optimal antiserum concentration of 1:1 500.