›› 2014, Vol. 57 ›› Issue (3): 308-314.doi:

• 研究论文 • 上一篇    下一篇


谷琳珠, 张传溪*   

  1. (浙江大学昆虫科学研究所, 杭州 310058)
  • 出版日期:2014-03-20 发布日期:2014-03-20

Modification and use of fp25k gene from Autographa californica multicapsid nucleopolyhedrovirus in stably transforming insect cell line Sf9

GU Lin-Zhu, ZHANG Chuan-Xi   

  1. (Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China)
  • Online:2014-03-20 Published:2014-03-20

摘要: 【目的】苜蓿丫纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)在昆虫细胞中连续传代以后,会出现从多多角体表型到少多角体表型的转变,这种转变与一个编码25 kDa蛋白的基因(few polyhedra, fp25k)突变失活有关。杆状病毒的fp25k基因突变后产生的包涵体(多角体)衍生病毒粒子变少而出芽型病毒粒子增加,会降低外源基因在杆状病毒表达系统中的表达。本研究拟改造fp25k并构建能持续表达FP25K蛋白的转基因昆虫细胞,以克服杆状病毒, fp25k基因易突变导致的表达系统缺陷。【方法】本实验通过改造杆状病毒, fp25k基因在细胞传代过程中容易产生突变的位点,得到 mfp25k,并将mfp25k构建到pIZT/V5-His载体上,重组载体转染Sf9细胞,通过Zeocin抗性筛选逐步淘汰未成功转化的Sf9细胞。【结果】成功改造AcMNPV的, fp25k基因的TTAA位点,得到pIZT-mfp25k重组载体。重组载体成功转染Sf9细胞,通过Zeocin抗性筛选后获得基因组中带有mfp25k的Sf9-mfp25k稳定的转基因细胞系。用AcMNPV的fp25k突变型病毒AcP2感染转基因Sf9-mfp25k昆虫细胞系与正常Sf9细胞,发现转基因Sf9-mfp25k昆虫细胞系表达的FP25K蛋白可弥补病毒, fp25k基因突变的缺陷。【结论】建立的Sf9-mfp25k转基因昆虫细胞系通过细胞表达FP25K蛋白,可以弥补因杆状病毒fp25k基因突变产生的缺陷。研究结果为构建稳定的杆状病毒昆虫细胞表达系统提供了新途径。

关键词: AcMNPV; fp25k, fp25k 修饰, 转基因昆虫细胞, 杆状病毒表达系统

Abstract: 【Aim】 After serial passages in some permissive insect cells, phenotype of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) has the potential to change from many polyhedra (MP) to few polyhedra (FP). This phenomenon is related to mutations occurred in a gene named fp25k which codes a 25 kDa protein. The AcMNPV fp25k mutants produce less occlusion-derived viruses (ODVs) and more budded viruses (BVs) than the wild type, usually resulting in reduction of foreign protein expression in the baculovirus expression vector system. To overcome the deficiency resulted from the mutations of fp25k in baculovirus expression vector system, we modified fp25k and constructed a transgenic insect cell line expressing FP25K protein. 【Methods】 The AcMNPV fp25k gene was modified in a site which is easy to be mutated during passage, and a modified fp25k, named mfp25k, was produced in this study. mfp25k  was combined to pIZT/V5-His vector, the recombinant vector was further used to transfect the Spodoptera frugiperda cells (Sf9), and Sf9 cells which were not successfully transformed were eliminated gradually by Zeocin selection. 【Results】 We successfully modified AcMNPV fp25k in TTAA site and got recombinant vector pIZT/V5-mfp25k. After transforming Sf9 cells and selecting with Zeocin, we finally got stable transgenic Sf9 cell line, Sf9-mfp25k, which contains mfp25k gene in the genome. Sf9-mfp25k  transgenic cells and normal Sf9 cells were both infected with AcP2, the AcMNPV fp25k mutant. The results indicated hat the transgenic Sf9-mfp25k cells expressing FP25K protein could rescue the phenotype of AcMNPV with fp25k mutations. 【Conclusion】 The transgenic Sf9-mfp25k  cells expressing FP25K protein could make up for the shortage of baculovirus caused by fp25k mutations. Our results provide a novel way to build stable baculovirus-insect cell expression system.

Key words: AcMNPV, fp25k; modified fp25k, transgenic insect cells, baculovirus expression vector system