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用RT-PCR方法,从采自重庆奉节的‘宫本’柑橘的2个样品中扩增出温州蜜柑萎缩病毒(Satsuma dwarf virus,SDV)SDV RNA2的3′末端,长度为975bp,与报道的SDV S-58 3′末端序列同源性分别为98.7%和98.4%。根据获得的序列设计引物,以含有SDV FJ 3′末端序列的质粒为模板,PCR扩增获得大小为654bp的SDV-FJ小外壳蛋白(Small coat protein,CPS)基因产物。构建了CPS与GST融合表达载体PGEX-CPS,在37℃、1.0 mmol·L-1IPTG条件下诱导表达,SDS-PAGE电泳分析表明成功表达出分子量约为42kD的GST-CP融合蛋白。以表达的融合蛋白为抗原免疫家兔,制备的抗血清的效价为1/12 800。用获得的抗体进行组织印迹分析表明,SDV在柑橘叶柄基部韧皮部维管束区域含量最高。
The 3 ’end of SDV RNA2 of Satsuma dwarf virus (SDV) was amplified by RT-PCR from two samples of’ Miyamoto ’citrus collected from Fengjie, Chongqing, with a length of 975 bp. The 3 ’terminal sequence homology of SDV S-58 was 98.7% and 98.4%, respectively. According to the designed primers, the SDV-FJ Small Coat Protein (CPS) gene product with a size of 654bp was obtained by PCR amplification using the plasmid containing the 3 ’terminal sequence of SDV FJ as a template. The recombinant expression vector pGEX-CPS was constructed and expressed under the condition of 37 ℃ and 1.0 mmol·L-1 IPTG. SDS-PAGE analysis showed that GST-CP fusion protein with a molecular weight of about 42 kD was successfully expressed. The expressed fusion protein was used as antigen to immunize rabbits. The antiserum titer was 1/12 800. Histoprinological analysis of the obtained antibodies showed that the SDV content was highest in the vascular bundles of phloem in the basal part of citrus petioles.