甘薯褪绿斑病毒外壳蛋白的分子变异及其特异抗血清制备  

Molecular variability of coat protein gene of Sweet potato chlorotic fleck virus and preparation of specific antiserum against CP

作　　者：乔奇[1,2,3] 秦艳红[1,2,3] 张德胜[1,2,3] 田雨婷[1,2,3] 王爽[1,2,3] 王永江[1,2,3] 张振臣[1,2,3] 

机构地区：[1]河南省农业科学院植物保护研究所,郑州450002 [2]河南省农作物病虫害防治重点实验室,郑州450002 [3]农业部华北南部作物有害生物综合治理重点实验室,郑州450002

出　　处：《植物病理学报》2014年第6期634-640,共7页Acta Phytopathologica Sinica

基　　金：国家甘薯产业技术体系建设项目(CARS-11-B-07);河南省农业科学院财政预算项目资助

摘　　要：甘薯褪绿斑病毒（Sweetpotato chlorotic fleckvirus，SPCFV）是侵染甘薯的主要病毒之一。本研究利用RT—PCR方法克隆了SPCFV中国4个分离物的外壳蛋白（CP）基因。序列分析表明，印基因全长900bp，编码299个氨基酸残基。4个分离物印基因的核苷酸序列一致性为78．3％-89．9％，推导的氨基酸序列一致性为91．3％-95．7％，存在较大的分子变异。不同分离物cP氨基酸序列N末端的第3-32位氨基酸为多变区。将四川分离物的印基因克隆到原核表达载体pET-28a（＋）上，SDS-PAGE分析表明，经IPTG诱导，印基因在大肠杆菌BL21（DE3）中得到了高效表达。以表达的蛋白为抗原免疫家兔，制备了SPCFVCP的特异性抗血清。ACP-ELISA检测结果表明，制备的抗血清效价达1：128000，可用于田间甘薯样品的检测。Sweet potato chlorotic fleck virus （SPCFV） is one of major viruses infecting sweet potato. The coat protein （CP） gene of SPCFV from four Chinese isolates were cloned by RT-PCR. Sequence analysis showed that the full-length cp gene comprised 900 nt and encoded 299 amino acid residues. The cp of the four isolates shared 78.3% -89.9% and 91.3%-95.7% identities at the nucleotide and amino acid level respectively. The third to 32nd amino acids of CP from different isolates were more variable compared with other regions. The cp of Sichuan isolate was cloned into expression vector pET-28a （＋） for overexpression in prokaryotic cells. The result of SDS-PAGE showed that a 36. 5 kDa specific fusion protein was produced after induction with IPTG. The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. The ELISA titer of the antiserum was 1 ： 128 000 in infected leaves supernatant. The antiserum was used for specific detection of SPCFV from field samples of sweet potato by ACP-ELISA.

关 键 词：甘薯褪绿斑病毒 外壳蛋白 分子变异 原核表达 抗血清制备 

分 类 号：S432.41[农业科学—植物病理学]