舞毒蛾几丁质酶基因的真核表达与重组病毒的获得

Eukaryotic expression of the chitinase gene in Lymantria dispar and construction of a recombinant baculovirus

几丁质是昆虫外骨骼和围食膜的重要组成部分,鉴于几丁质酶在昆虫生长发育过程中发挥着举足轻重的作用,应用昆虫几丁质酶为探索新的生物防治害虫的方法提供了途径。本文分别根据苜蓿银纹夜蛾Autographa californica核型多角体病毒多角体蛋白基因序列和编码舞毒蛾Lymantria disparⅠ型几丁质酶基因的开放阅读框设计引物,使用聚合酶链反应扩增出以上两个基因,全长分别为783 bp和1 737 bp。构建重组质粒pFastBac-LdCht和pFastBac-AcPH-LdCht,转化大肠杆菌DH10Bac后获得重组穿梭载体,通过脂质体介导转染Sf9细胞产生重组杆状病毒AcMNPV-AcPH--LdCht和AcMNPV-LdCht,分别用于表达蛋白和获得重组病毒。细胞成功表达出有活性的舞毒蛾几丁质酶,并在棉铃虫体内扩增得到重组病毒。研究为深入了解昆虫几丁质酶性质提供依据,并为应用重组病毒奠定基础。

Chitin is an important part of exoskeletons and peritrophic membrane of insect. In view of chitinase playing a pivotal role in the process of insect development, application of insect chitinase provides a strategy for exploring new methods for biological control of pests. In this paper, based on Autographa californica nuclear polyhedrosis virus polyhedrin gene sequence and the open reading frame of I chitinase gene from Lymantria dispar couples of primers were designed to clone the above two genes, with total length of 783 bp and 1 737 bp, respectively. Recombinant plasmid pFastBac-LdCht and pFastBac-AcPH-LdCht were constructed and transformed into E. coli DH10Bac to form recombinant bacmids which then were transfected into Sf9 cells to produce recombinant baculovirus AcMNPV-AcPH--LdCht and AcMNPV-LdCht. They were used to express the protein and get recombinant virus respectively. Cells successfully expressed active chitinase and amplified recombinant viruses in the Helicoverpa armigera. This study provided the basis for in-depth understanding of the insect chitinase and a potential way for recombinant virus application.