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

园艺学报 ›› 2012, Vol. 39 ›› Issue (1): 64-72.

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

武改霞1，李婷婷1，孙现超1,2,*，青玲1

（1西南大学植物保护学院，植物病害生物学重庆市高校级重点实验室，重庆 400716；2国家柑桔工程技术研究中心，中国农业科学院柑桔研究所，重庆 400712）

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-01-25 发布日期:2012-01-25
通讯作者: 孙现超

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

WU Gai-xia1，LI Ting-ting1，SUN Xian-chao1,2,*，and QING Ling1

（1Chongqing Key Laboratory of Plant Disease Biology，College of Plant Protection，Southwest University，Chongqing，400716，China；2 National Center of Citrus Engineering and Technology Research，Citrus Research Institute，Chinese Academy of Agricultural Sciences，Chongqing，400712，China）

Received:1900-01-01 Revised:1900-01-01 Online:2012-01-25 Published:2012-01-25
Contact: SUN Xian-chao

摘要/Abstract

摘要： 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。

关键词: 柑橘, 温州蜜柑萎缩病毒, 小外壳蛋白, 原核表达, 抗体

Abstract: 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.

Key words: citrus, Satsuma dwarf virus, small coat protein, prokaryotic expression, antiserum

中图分类号:

武改霞;李婷婷;孙现超;;青玲 . 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[J]. 园艺学报, 2012, 39(1): 64-72.

WU Gai-xia;LI Ting-ting;SUN Xian-chao;;and QING Ling. Cloning of the Small Coat Protein Gene，Prokaryotic Expression and Antiserum Preparation of Satsuma dwarf virus[J]. ACTA HORTICULTURAE SINICA, 2012, 39(1): 64-72.

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