温州蜜柑萎缩病毒小外壳蛋白基因克隆与原核表达及其抗体制备

 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975 bp，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 654 bp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-1 IPTG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42 kD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。

Cloning of the Small Coat Protein Gene,Prokaryotic Expression and Antiserum Preparation of Satsuma dwarf virus

The 3′terminal of SDV(Satsuma dwarf virus)RNA2 was amplified from two plants of Miyamoto Satsuma mandarin from Fengjie in Chongqing by RT-PCR.The amplified fragments are 975 bp in size and share 98.7%and 98.4%nucleotide identities with that of SDV S-58,respectively.The small coat protein(CPS)gene with a size 654 bp of SDV-FJ was amplified from the plasmid containing the SDV RNA2 3′terminal by PCR and cloned into the prokaryotic expression vector of PGEX-6p-1 to construct a recombinant vector named PGEX-CPS.The recombinant plasmid was transformed into E.coil BL21 to express the GST-CPS protein in the optimized condition.GST-CPS protein was purified to immunize rabbit for preparing anti-CPS antibody.The results showed that the 42 kD GST-CPS protein was successfully expressed in E.coil BL21 induced with 0.3 mmol·L -1 IPTG at 28℃.Anti-CPS antibody with the title of 1/12 800 was obtained from the rabbit immunized with the purified GST-CPS protein.The result of tissue bolt with the anti-CPS antibody showed that the base of petiole contain more SDV than other tested tissues of infected Miyamoto Satsuma mandarin.