甘蔗黄叶病毒P0基因的克隆与原核表达及抗血清制备

根据已发表ScYLV-P0基因系列设计特异性引物,应用RT-PCR技术从甘蔗病叶的mRNA扩增得到目的DNA片段.以pET32a（＋）为原核表达载体,构建重组表达质粒pET32a-P0.经过双酶切鉴定和DNA测序后,将重组表达质粒转入大肠杆菌BL21 （DE3）pLySs,在30℃培养条件下IPTG诱导表达.通过SDS-PAGE电泳检测融合蛋白表达情况.表达结果显示,在该表达系统中,融合表达蛋白P0是以包涵体形式的蛋白存在;P0融合蛋白大小约45kDa,与P0开放阅读框的理论推算值29.991 kDa加上载体自身蛋白约18 kDa相符,用Ni2＋-NTA琼脂糖亲和层析纯化融合蛋白,免疫家兔制备出抗血清,通过酶联法（ID-ELISA）测定本实验制备的ScYLY-P0抗血清工作浓度为1：25000.

Cloning and Prokaryotic Expression of Sugarcane Yellow Leaf Virus P0 Gene and Preparation of Antiserum

A specific primers used in RT-PCR were designed according to the sequences of SCYLV-P0 gene published.A target fragment of 190 was isolated by RT-PCR with RNA isolated from diseased leaves as template. The complete SCYLV-P0 gene was taken according to the sequence analysis. The plasmid of pET32a （＋） was taken as vector bone, and the prokaryo expression plasmids named as pET32a-P0 with the target gene of 190 was constructed. The recombinant plasmids pET32a-P0 were tested by double digestion tests and sequncing, then they were transformed into competent cells of E. coli BL21 （DE3） pLySs and induced by IPTG to express the target gene. The P0 fusion protein was expressed as inclusion body, the molecular weight of P0 fusion protein was about 45 kDa with two parts of 29.991 kDa according to P0 and 18 kDa according to pET32a（＋）. The fusion protein was then purified with Ni2 ＋-NTA agarose affinity chromatography, and used to immune rabbits for antiserum preparation. The optimal titer of the antiserum was determined to be as 1：25000 by indirect enzyme-linked immunosorbent assay （ID-ELISA）.