凡纳滨对虾LvRab5B蛋白与IHHNV病毒蛋白的互作

为了探究LvRab5B蛋白在凡纳滨对虾抗病毒感染中的作用,实验分别构建了LvRab5B蛋白在昆虫和酵母细胞中的融合表达载体,将不同的载体导入不同的细胞中,利用免疫荧光和酵母双杂交的方法研究了Lv Rab5B蛋白在昆虫细胞中的表达以及Lv Rab5B蛋白与病毒IHHNV之间的互作关系;通过qRT-PCR方法研究了该蛋白在健康对虾不同组织中的表达情况以及凡纳滨对虾分别感染IHHNV和WSSV后不同时间点的相对表达量。结果显示,Lv Rab5B基因融合蛋白能够在昆虫细胞中表达;Lv Rab5B蛋白与IHHNV病毒衣壳蛋白CP无相互作用,而与非结构蛋白NS1相互作用明显,与非结构蛋白NS2作用较弱。qRT-PCR结果显示,LvRab5B基因在凡纳滨对虾心脏、鳃腺、肠道、胃、肝胰脏和肌肉中都表达,在肠道中表达量最高,肝胰脏次之;Lv Rab5B蛋白在凡纳滨对虾机体感染病毒前后的表达情况不同,感染初期表达降低,随后迅速上升,末期下降。研究表明,LvRab5B基因参与凡纳滨对虾抵抗IHHNV和WSSV病毒的先天免疫过程,为进一步研究Lv Rab5B蛋白在对虾机体中的免疫功能及作用机制奠定了基础。

Interaction between Litopenaeus vannamei Rab5B protein (LvRab5B) and IHHNV proteins

To study the antiviral function of LvRab5B in the virus resistance of Litopenaeus vannamei, we constructed the fusion expression vectors of LvRab5B protein in insect cells and in yeast cells, respectively; studied the expression of LvRab5B protein in insect cells using immunofluorescence technique; studied the interaction between LvRab5B protein and IHHNV proteins using yeast two-hybrid technique; studied the expression of LvRab5B in different tissues of normal L. vannamei and the relative expression of LvRab5B at different time points after infection in L. vannamei infected with IHHNV or WSSV using qRT-PCR technique. The results showed that the fusion LvRab5B protein was able to express in insect cells. LvRab5B protein had no interaction with capsid protein (CP) of IHHNV, while it interacted with nonstructural protein (NS1) of IHHNV, and was weakly interacted with nonstructural protein (NS2) of IHHNV. qRT-PCR showed that LvRab5B was expressed in the heart, gills, intestine, stomach, hepatopancreas and muscle of normal L. vannamei, and the expression was the highest in the intestine, followed by the hepatopancreas. The relative expression of LvRab5B was different after viral infection. The expression decreased at the initial stage of infection, then increased rapidly, finally decreased at the end stage. This study shows that LvRab5B is involved in the innate immune process of L. vannamei against IHHNV or WSSV infection, which lays a foundation for further studies of the immune function and mechanism of LvRab5B protein in shrimp.