家蚕浓核病毒中国株非结构蛋白1(NS1)的表达

Expression of non-structural protein 1 of Bombyx mori densovirus-3

摘要:

利用PCR技术扩增出BmDNV-3 NS1基因, 将目的基因与原核表达载体pET-30a进行连接, 转化BL21 star菌并在该菌中表达, 经Western blot鉴定表达的产物为BmDNV-3 NS1蛋白, 纯化NS1蛋白并制备兔多克隆抗体。同时BmDNV-3 NS1基因亚克隆到杆状病毒转移载体pFastBac-HTb-eGFP中, 转化BmDH10BAC感受态细胞, 提取的重组Bacmid通过脂质体包埋转染家蚕BmN细胞, 再以收获的重组病毒感染家蚕幼虫。家蚕BmN细胞和幼虫感染重组病毒2d后均观察到绿色荧光, 经SDS-PAGE分析真核表达的产物与预测的NS1-eGFP融合蛋白大小不一致, 说明NS1-eGFP融合蛋白被昆虫内源性的蛋白酶降解。降解的产物用NS1蛋白抗体进行Western blot鉴定为BmDNV-3 NS1蛋白。

Abstract:

We cloned the NS1 gene of BmDNV-3 into the prokaryotic expression vector pET-30a after we amplified the NS1 gene by PCR, and then we transformed the pET-30a-NS1 plasmid into BL21 star to express BmDNV-3 NS1. After we induced NS1 expression by IPTG, we used Western blot analysis to indentify the recombinant protein, the result indicated that the recombinant protein was BmDNV-3 NS1. After purification, we used NS1 to immunize New Zealand white rabbits following standard protocol to harvest anti-NS1 anti serum. On the other hand, we cloned the BmDNV-3 NS1 into pFastBacHTb-eGFP vector, and then transformed the pFastBacHTb-NS1-eGfp into BmDH10BAC to harvest recombinant bacmid genome. We obtained the recombinant virus from the cells, which was transfected by the recombinant bacmid genome using liposomes. We used the virus genome to infect Bombyx mori larvae. We observed the fluorescence in the cells and silkworm larvae at 2 days post infection, and then we used SDS-PAGE and fluorescence image analysis to identify the fusion protein. The result showed that the size of the fusion protein was not consistent with the expected size of NS1-eGFP, indicating the fusion protein was degraded randomly by the intrinsic digestive protease. To further confirm the fusion protein, we used Western blot with an anti-NS1 antibody.