对虾白斑综合征病毒囊膜蛋白VP110在中国明对虾鳃细胞中结合蛋白的鉴定与特性

Interaction between WSSV envelope protein VP110 and binding proteins on the gills of Chinese shrimp, Fenneropenaeus chinensis

为了鉴定对虾白斑病综合征病毒(WSSV)囊膜蛋白VP110在中国明对虾(Fenneropenaeus chinensis)鳃细胞中的结合蛋白,运用pET-32(a)+载体构建了1段含RGD模体的截短VP110原核重组表达质粒,转化大肠杆菌诱导表达后获得分子量为41 kD的截短重组VP110蛋白(rVP110)。以rVP110作为诱饵蛋白,运用pull-down实验结合蛋白质谱分析鉴定rVP110结合蛋白,结果显示,中国明对虾鳃细胞中的肌动蛋白和精氨酸激酶(arginine kinase,AK)与rVP110具有结合作用。利用PCR扩增中国明对虾AK编码基因,将其与表达载体pGEX-4T-1连接后转化大肠杆菌诱导表达获得重组AK蛋白(rAK),通过pull-down实验进一步证实rAK可与rVP110发生结合。克氏原螯虾(Procambarus clarkia)体内中和实验结果显示,rAK对WSSV感染克氏原螯虾具有一定的中和作用,能延缓螯虾的死亡进程。另外,中国明对虾在人工感染WSSV后,荧光定量PCR检测结果显示,AK基因表达水平显著上调,18 h时达到峰值,然后下降至正常水平;酶底物法检测结果同样显示,鳃细胞中AK酶活性在感染WSSV后发生显著上调。本研究旨在为深入了解WSSV囊膜蛋白VP110在WSSV感染宿主过程中的作用提供基础依据。

White spot syndrome virus （WSSV） is one of the most virulent viral pathogens of the shrimp-farming indus- try worldwide, causing high mortality and resulting in serious economic losses. The viral envelope proteins play very important roles in WSSV infection of shrimps. To investigate the interaction between WSSV envelope protein VP110 and gill cell proteins of the Chinese shrimp, Fenneropenaeus chinensis, a truncated VP110 protein containing the RGD motif was recombinanfly expressed in Escherichia coli BL21（DE3） as a fusion protein with Trx/His/S-tag. Using a pull-down assay, two prominent protein bands with molecular weights of 40 kD and 42 kD were identified from gill cell proteins ore chinensis using recombinant Vial 10 （rVP110）, which were identified as arginine kinase （AK） and/^-actin by mass spectrometry （MS） analysis. The AK gene of E chinensis was cloned and expressed as a fusion protein with GST-tag using the pGEX-4T-1 vector, and the binding interaction between the recombinant AK （rAK） and rVP110 was further confirmed by a pull-down assay. In an in vivo neutralization assay, rAK appeared to be able to partially block WSSV infection and delayed the death of WSSV-infected crayfish. In addition, following WSSV infection, AK mRNA level in the gills was upregulated compared with the control group, as assessed by real-time quantitative PCR. The AK enzyme activity in the gills was also upregulated. These results suggested that AK in E chinensis plays a role in WSSV infection.