斜纹夜蛾核多角体病毒p49基因在大肠杆菌中的表达及其在昆虫细胞中的表达时相分析

Expression of SpltMNPV p49 Gene in Escherichia coli and Determination of The Time Course of Its Expression in Insect Cells

利用PCR方法从斜纹夜蛾核多角体病毒 (SpltMNPV)基因组中扩增获得了细胞凋亡抑制基因p4 9的完整ORF并将其克隆于pMD1 8 T载体 ,其序列分析结果与文献报道一致。将基因重组于硫氧还蛋白融合表达载体pThioHisC ,在大肠杆菌BL2 1 (DE3)中获得了稳定表达 ,表达的P4 9融合蛋白占菌体总蛋白 30 %左右 ,主要以包涵体形式存在。分离纯化重组表达的SpltMNPVP4 9蛋白作为抗原 ,免疫家兔制备得到效价高于 1∶1 0 0 0 0的抗重组P4 9蛋白多克隆抗体。应用制备的抗体对受SpltMNPV感染的Sl细胞中P4 9蛋白的表达时相进行分析 ,结果显示P4 9蛋白在细胞感染后 3h内便可检测到 。

The apoptotic suppressor p49 gene was amplified by PCR from the genome of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) and cloned into the pMD18-T vector. Sequence analysis verified the cloned fragment. The gene was then inserted into thioredoxin fusion expression vector pThioHis C and expressed stably in E.coli BL21(DE3) by induction with IPTG. The expression products were about 30% of the total bacteria proteins and exsisted as inclusion bodies. Specific antibodies with a high titre of over 1:10000 were obtained by immunizing rabbits with the purified fusion protein and was used to determine the time course of P49 expression in SpltMNPV-infected Sl cells. The results showed that P49 expression startups within 3 hours post infection and remains low level during the whole course of infection.