杆状病毒核心基因ac78的克隆、表达与活性研究

Cloning and Expression of Baculovirus Core Gene ac78 in Escherichia coli and Determination on Its Enzyme Activity

杆状病毒核心基因ac78在杆状病毒的生活周期中具有重要作用。生物信息学分析发现,Ac78与斯氏假单胞菌的细胞色素C氧化酶cbb3-型亚基III具有一定的序列一致性,表明Ac78可能具有氧化酶活性,并在氧化还原反应中具有一定功能。用PCR的方法成功扩增得到ac78基因,该基因序列与GenBank上的苜蓿丫纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV) ac78基因的核苷酸同源性100%,将其克隆至原核表达载体p QE30,构建了ac78基因原核表达质粒,转化大肠杆菌M15[pREP4],在1 mmol/L IPTG和低温诱导下超量表达了可溶性的目的蛋白。使用Ni-NTA Resin蛋白纯化树脂获得了较纯的含有6×His接头的Ac78蛋白,使用纯化细胞色素C氧化酶活性测定试剂盒测定其细胞色素C氧化酶活性,结果为阴性,表明Ac78可能不具有细胞色素C氧化酶活性。上述研究结果为深入探究Ac78的作用机理打下了坚实的基础。

Autographa californica multiple nucleopolyhedrovirus(AcMNPV)ORF78(ac78)is one of baculovirus core genes.It plays an important role in the baculovirus life cycle.Sequence analysis identified that Ac78 had the sequence characteristics of cytochrome C oxidase.To define the mechanism of Ac78,the ORF of ac78 gene was obtained by PCR method from AcMNPV genomic DNA.The PCR product was cloned into the expression vector pQE30 and transformed into M15[pREP4],Ac78 was expressed after the induction with IPTG(Isopropylthio-β-D-galactoside)at 16℃for 20 h.The fusion protein was purified by Nickel metal-affinity resin column chromatography.Determination of cytochrome C oxidase activity showed that there was no oxidase activity detected in Ac78,suggesting that Ac78 might not have cytochrome C oxidase activity.This lays a foundation for further research of the mechanism of Ac78 in the course of baculovirus infection.