芜菁花叶病毒长春分离物p3基因的克隆、序列分析及P3蛋白抗血清的制备  

Cloning,sequencing of p3 gene of Turnip mosaic virus isolates in Changchun and preparation of P3 protein antiserum

作　　者：张雅琦[1] 祝富祥 王凤婷[1] 潘洪玉[1] 李向东[2] 刘金亮[1] 

机构地区：[1]吉林大学植物科学学院,长春130062 [2]山东农业大学植物保护学院,泰安271018

出　　处：《植物病理学报》2015年第6期585-591,共7页Acta Phytopathologica Sinica

基　　金：国家自然科学基金项目(31201485);高等学校博士学科点专项科研基金(20100061120036;20123702110013)

摘　　要：用RT-PCR方法从长春感染芜菁花叶病毒（ Turnip mosaic virus,TuMV）的十字花科蔬菜中扩增获得该病毒的p3基因,并对其序列进行了比较分析。结果表明,本研究所获得的10个TuMV分离物p3基因含1 065个核苷酸,其序列一致率为98.8%～99.6%,与GenBank中其他15个TuMV分离物核苷酸一致率为80.9%～99.4%。根据p3基因核苷酸序列构建的系统进化树显示：25个TuMV分离物可分为4个组,本研究得到的10个TuMV分离物均属于basal-BR组。将TuMV JCR06分离物p3基因N端663 bp片段克隆至原核表达载体pET-28a（＋）,并在大肠杆菌BL21（DE3） pLysS中表达出分子量约为28 kDa的融合蛋白。以纯化的融合蛋白为抗原免疫家兔,制备了P3蛋白的特异性抗血清。以TuMV侵染的萝卜为抗原,间接ELISA测定抗血清的效价为1∶2 048。Western blotting分析表明,制备的抗血清能与诱导表达的融合蛋白发生特异性反应。Ten Turnip mosaic virus （TuMV） isolates were collected from infected cruciferous vegetables in Changchun. The p3 genes of these 10 isolates consisted of 1 065 nucleotides, with identities of 98.8%-99.6% at the nucleotide level. These p3 genes shared nucleotide identities of 80.9%-99.4% with other 15 TuMV isolates available in the GenBank. In the phylogenetic tree constructed with the complete nucleotide sequences of the p3 genes, the 25 TuMV isolates were divided into 4 groups, and the 10 TuMV isolates characterized in this resarch belonged to group basal-BR. The N-terminnal 663 nucleotides of p3 gene in isolate JCR06 was cloned into expression vector pET-28a（＋） and transferred into E. coli BL21（DE3） pLysS. SDS-PAGE showed that the N-terminnal fragment of p3 gene was expressed as a 28 kDa fusion protein with IPTG induction. Rabbit was immunized with purified fusion protein to obtain the antiserum with high specificity. The titer was 1∶2 048 determined by indirect ELISA test to detect TuMV in radish. Western blotting showed that the prepared antiserum could specifically bind to fusion protein.

关 键 词：芜菁花叶病毒 p3基因 序列分析 原核表达 抗血清制备 

分 类 号：S432.41[农业科学—植物病理学]