杆状病毒SpltMNPV gp41基因的克隆、表达及抗体制备

摘要：GP41是杆状病毒包埋型病毒粒子（Occlusion-derived virus，ODV）特有的糖蛋白，它介导芽殖型病毒粒子（Budded virus，BV）的核衣壳通过胞核。用PCR方法扩增得到斜纹夜蛾核多角体病毒（Spodoptera litura nucleopolyhedro virus，SpltMNPV）gp41基因, 并将其克隆至5′端有6×His编码序列的原核表达载体pQE30上，转化大肠杆菌(Escherichia coli )M15pREP4]，在1 mmol/L异丙基-D-硫代半乳糖（IPTG）诱导下超量表达了与理论预测值相符的1个37.9 kD融合蛋白。以纯化的融合蛋白为抗原，免疫新西兰大白兔制备了特异的抗GP41抗体。Western印迹分析表明，该抗体适合进一步分析SpltMNPV GP41蛋白。

Cloning and Expression of Spodoptera litura nucleopolyhedro virus gp41 Gene in Escherichia coli and Preparation of Its Antibody

Abstract: GP41, a major glycoprotein, has been identified in the Occlusion-derived virus（ODV）of baculoviruses, which was required for the egress of nucleocapsids from the nucleus in the pathway of the Budded virus（BV）synthesis. The ORF of Spodoptera litura nucleopolyhedro virus（SpltMNPV）gp41 gene was obtained by PCR method from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid（pT-gp41）. The gp41 gene was recombined in vitro with prokaryotic expression vector pQE30 and transformed into Escherichia.coli M15 pREP4]. The M15 pREP4] strain, containing gp41 recombinant plasmid, expressed a 37.9 kD 6×His-tag fusion protein after induction with 1 mmol/L IPTG (isopropylthio-β-D-galactoside). The fusion protein was purified with Ni-NTA resin column and used as the immunogen to raise a GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.