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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1/4 096。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。
VPg gene was amplified from maize leaves infected with Beijing sugarcane mosaic virus (SCMV-BJ) by RT-PCR and the VPg gene was ligated into prokaryotic expression vector pET-28a. The obtained recombinant was transformed into E. coli BL21 (DE3) and induced with IPTG. SDS-PAGE analysis showed that VPg was highly expressed in E. coli, resulting in a fusion protein with a molecular weight of 30 kD. The fusion protein was purified and immunized rabbits to obtain a specific high antiserum. The antiserum titer of ACP-ELISA was 1/4 096. Immunogold labeling of maize stems, veins and leaves infected by healthy maize and virus was performed using the antiserum. The results showed that gold particles were located in the cell wall of phloem sieve of stem and vein.