杆状病毒AcMNPV ac2 基因功能研究.doc

  杆状病毒 AcMNPV ac2 基因功能研究# 汪敏，王瑾雯，邓日强，王珣章** （中山大学生命科学学院，广州 510275） 5 10 15 20 25 30 35 40 45 摘要：bro（baculovirus repeat orf）基因是一类普遍存在于杆状病毒基因组中的重复基因， 它们在不同基因组间的长度、相对位置、拷贝数甚至氨基酸序列上都存在较大差异，保留一 个如此多态性的重复基因对于力求精简的杆状病毒基因组而言令人费解。本论文开展了对 AcMNPV 中的 bro 基因即 ac2 的功能研究。通过逆转录分析发现 ac2 基因的转录 8h 就可以 观察到，并持续到 96h。利用 ET recombination 系统构建了 ac2 基因缺失型 AcMNPV 重组 病毒，并且通过 Bac-to-Bac 系统构建了带有绿色荧光蛋白基因（ gfp）和多角体基因 （polyhedrin）双标记的野生型（Ac-GP）、ac2 基因敲除型（Ac-ac2-KO-GP）及 ac2 基因补 回型（Ac-ac2-REP-GP）重组病毒。将三种重组病毒转染离体培养昆虫细胞系 Sf9，通过荧 光显微镜观察、光学显微镜观察、病毒滴度测试、透射电镜观察实验，发现了 ac2 缺失后使 病毒滴度下降 100 倍左右，ODV 呈单核衣壳包被的形态，ac2 的缺失导致多角体数量的减少。 研究结果表明了 ac2 基因对于 BV 的增殖不是必须的，但是有微弱的促进作用，ac2 基因可 能会在 ODV 的包被过程中发挥一定作用。 关键词：杆状病毒；AcMNPV；ac2；ODV；重复基因 中图分类号：Q939.4 Functional Analysis of AcMNPV ac2 Gene WANG Min, WANG Jinwen, DENG Riqiang, WANG Xunzhang (Life Science School,Sun Yat-sen Univeristy, GuangZhou 510275) Abstract: This paper focused on the function of ac2 of AcMNPV, which is a bro gene. Bro(baculovirus repeat orf), a repeated gene commonly found in most baculovirus, shows great gene polymorphism as to its length, relative position, number of copies and even function in different genomes. Temporal transcription pattern of ac2 was described which displayed a typical late gene characteristic. Then the function analysis of ac2 by generation of ac2 deletion mutant virus with ET recombination and the Bac-to-Bac system in E coli was performed. Three recombinant viruses, ac2 deletion type (Ac-ac2-KO-GP), ac2 repair type (Ac-ac2-REP-GP), and wild type (Ac-GP) viruses were generated. Fluorescence microscopy showed that ac2 deletion had no apparent effect on spreading of BV. Optical microscopy showed that fewer OBs was observed in individual Sf9 cells transfected with ac2 deletion virus than wild type and ac2 repair virus.TCID50 assessment revealed a steady increase for ac2 defective virus titer but the ratio declined after 48 h.p.i., and BV yields was 100-fold lower at 96 h.p.i.. Electron microscopy observation exhibited single nuc