甘蔗花叶病毒VPg蛋白的原核表达及其在玉米中的免疫定位

 利用RT-PCR方法从感染甘蔗花叶病毒北京分离株(SCMV-BJ)的玉米叶片中扩增得到VPg基因,将VPg基因连接到原核表达载体pET-28a上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,VPg在大肠杆菌中获得了高效表达,产生的融合蛋白分子量为30 kD。将融合蛋白纯化后免疫兔子获得了特异性较高的抗血清。ACP-ELISA测定抗血清的效价为1/4096。利用该抗血清对健康玉米和病毒侵染的玉米茎、叶脉和叶片进行免疫金标记,结果表明在茎和叶脉的韧皮部筛管的细胞壁处有金颗粒。

Prokaryotic expression and subcellular immuno-localization in maize of the VPg protein of Sugarcane mosaic virus

The VPg gene of Sugarcane mosaic virus Beijing isolate (SCMV-BJ) was amplified by RT-PCR from maize (Zea mays) leaves infected by SCMV-BJ, and cloned into expression vector pET-28a. The re-combinant plasmid was transformed into E. coli BL21(DE3)and induced by IPTG to express fusion protein. The result of SDS-PAGE analysis showed that the VPg was highly expressed after IPTG induction. The molecular weight of the fusion protein was about 30 kD. The highly specific antiserum was produced after rabbit was immunized with purified fusion protein, and the titer of the obtained antiserum was 1:40% determined by antigen-coated plate-ELISA. To determine the localization of the protein in maize cells, the stem, vein and leaf tissues collected from SCMV-infected or healthy maize plants were examined by immuno-gold labeling technique. The results indicated that this protein was located in the phloem cell walls of SCMV-infected stem and veins.