芜菁花叶病毒P3基因的克隆、原核表达及抗血清制备.docVIP

  芜菁花叶病毒 P3 基因的克隆、原核表达及 抗血清制备# 张雅琦1，王凤婷1，祝富祥1，潘洪玉1，李向东2，刘金亮1** 5 10 15 20 25 30 35 40 （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） 摘要：用 RT-PCR 方法从感染芜菁花叶病毒( Turnip mosaic virus，TuMV)的十字花科蔬菜中 扩增获得该病毒的 P3 基因，并对其序列进行了分析。结果表明，本研究所获得的 10 个 TuMV 分离物 P3 基因编码区均含有 1065 个核苷酸，核苷酸同源性为 98.8~99.6%，与 GenBank 中 其他 15 个 TuMV 分离物核苷酸同源性为 80.9~99.4%。根据完整 P3 基因核苷酸序列构建系 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 基因克隆到原核表达载体 pET-28a(+)，并在大 肠杆菌 BL21(DE3) pLysS 中表达出分子量为 28kDa 的融合蛋白。以纯化的重组蛋白为抗原 免疫家兔制备 P3 蛋白的多克隆抗体，间接 ELISA 测定抗血清的效价为 1/4096。Western blotting 分析表明，制备的抗血清能与重组融合蛋白发生特异性反应。 关键词：植物病理学；芜菁花叶病毒；P3 基因；序列分析；原核表达；抗血清制备 中图分类号：S432.4+1 Cloning, prokaryotic expression of P3 gene of Turnip mosaic virus and antiserum preparation ZHANG Yaqi1, WANG Fengting1, ZHU Fuxiang1, PAN Hongyu1, LI Xiangdong2, LIU Jinliang1 (1. College of Plant Sciences,Jilin University,Changchun 130062; 2. College of Plant Protection,Shandong Agricultural University,Taian 271018) Abstract: Ten isolates of Turnip mosaic virus (TuMV) were collected from infected brassica vegetables in Jinlin province. The P3 genes were amplified by RT-PCR, cloned and determined P3 genes of the 10 isolates were all composed of 1 065 nucleotides shared similarity of 98.8~99.6% at the nucleotide acid level. The obtained P3 gene sequences were compared with other 15 TuMV isolates available in the GenBank, and shared similarity of 80.9~99.4% at nucleotide acid level. The phylogenetic tree constructed with the complete nucleotide sequence of the P3 genes showed that 25 TuMV isolates were divided into 4 groups, and the 10 TuMV isolates belonged to group basal BR. The N-terminnal 663 nucleotides of P3 gene in JCR06 isolate was cloned into expression vector pET-28a(+) and transferred into E. coli BL21(DE3) pLysS. SDSshowed that P3 gene was expressed as a 28kDa fusion protein with IPTG induction. Rabbit was immunized with purified fusion protein to obtain the antiserum with high specificity. The t