杆状病毒Ac110蛋白的保守氨基酸位点N27对病毒在中肠建立有效感染起重要作用

发布时间：2018-11-12 17:30

【摘要】：杆状病毒是节肢动物,特别是鳞翅目昆虫的重要病原微生物,在生物防治中有着广泛的应用前景。其对昆虫虫体口服感染的过程中,需要一类称为杆状病毒口服感染因子的帮助才能在虫体中肠成功建立系统感染。Ac110是本实验室最新发现的口服感染因子之一,但其作用机制仍是未知。在本研究中,通过定点突变技术成功构建了两株Ac110保守氨基酸位点突变型重组杆状病毒,将病毒DNA转染昆虫细胞Sf9后,通过蛋白免疫印迹实验验证了Ac110的突变体蛋白均能够正常表达;接着通过病毒滴度测定实验证明了重组病毒均能在Sf9细胞中产生与补回型病毒相当的感染性病毒粒子,说明Ac110的这两个保守氨基酸位点的突变并不影响病毒在细胞中的复制和在细胞之间的传播;最后将纯化的病毒多角体口服感染甜菜夜蛾幼虫进行生物测定实验,发现Ac110的N27氨基酸位点的突变对昆虫幼虫的口服感染能力显著性降低,而L35氨基酸位点的突变则没有影响。
[Abstract]:Baculovirus is an important pathogenic microorganism of arthropods, especially Lepidoptera insects, and has a wide application prospect in biological control. In the process of oral infection of insect parasites, it needs the help of a class of oral infection factors called baculovirus in order to successfully establish a systemic infection in the midgut of insect. Ac110 is one of the newly discovered oral infection factors in our laboratory. But its mechanism is still unknown. In this study, two recombinant baculoviruses with conserved amino acid site mutation of Ac110 were successfully constructed by site-directed mutagenesis. The recombinant baculovirus DNA was transfected into insect Sf9 cells. The expression of Ac110 mutant proteins was confirmed by Western blot assay. Then the virus titer assay showed that the recombinant virus could produce the same infective virus particles as the reconstituted virus in Sf9 cells. These two conserved amino acid site mutations of Ac110 did not affect the replication of the virus in cells and the transmission between cells. Finally, the purified virus polyhedrosis was orally infected with beet Spodoptera exigua larvae. The results showed that the mutation of N27 amino acid site of Ac110 significantly decreased the oral infection ability of insect larvae, but the mutation of L35 amino acid site had no effect on insect larva oral infection ability.
【作者单位】：中山大学生命科学学院有害生物控制与资源利用国家重点实验室;江西科技师范大学生命科学学院;
【基金】：中央高校基本科研业务费专项资金(15lgjc20)
【分类号】：S476.13

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