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斜纹夜蛾核型多角体病毒Ⅱ型(SpltMNPVⅡ)分离株是繁殖率和毒力极强的新型病毒株,ORF63是该病毒分离株的一个功能未知基因。从SpltMNPVⅡ分离株基因组中克隆了ORF63。序列分析表明,该基因读码框为1 071 bp,编码356个氨基酸,蛋白质分子质量为41.3 kD;起始密码子ATG上游存在一个早期和晚期启动子基序,编码蛋白序列含有CNX、MutH等多种结构域。启动子活性和转录时相分析表明ORF63是一个早、晚期表达的基因,在病毒感染4 h和18 h时有2个转录峰,24 h以后转录水平略有下降,但总体趋于稳定。构建该基因的原核表达载体pET-28a-ORF63,并转化大肠杆菌BL21(DE3),表达并纯化融合蛋白后制备多克隆抗体,Western blot检测制备的多克隆抗体特异性较好,效价可达1∶3 200以上。由以上结果推测,SpltMNPVⅡ分离株的ORF63基因是一个早期和晚期均有表达的病毒基因,可能与SpltMNPVⅡ感染宿主细胞后自身DNA的复制有关,参与早期芽生病毒(BV)的发生和晚期包涵体衍生病毒(ODV)的成熟2个过程。
Spodoptera litura nuclear type II (SpltMNPVII) isolates are highly virulent strains and virulence strains, ORF63 is a function unknown gene of the virus isolates. ORF63 was cloned from the genome of SpltMNPVII isolates. Sequence analysis showed that the reading frame of this gene was 1 071 bp, encoding a protein of 356 amino acids with a molecular mass of 41.3 kD. There was an early and late promoter motif upstream of the ATG start codon and the protein sequence contained CNX, MutH, etc. A variety of domains. Promoter activity and transcriptional phase analysis showed that ORF63 was a gene expressed early and late, with two transcriptional peaks at 4 h and 18 h after virus infection. Transcription levels decreased slightly after 24 h, but overall stabilized. The prokaryotic expression vector pET-28a-ORF63 was constructed and transformed into E.coli BL21 (DE3). The fusion protein was expressed and purified to prepare polyclonal antibody. The specificity of the polyclonal antibody prepared by Western blot was good and the titer was up to 1: 3 200 or more. From the above results, it is speculated that the ORF63 gene of SpltMNPVII isolates is an early and late expression of the virus gene may be associated with SpltMNPVII host cells infected with their own DNA replication involved in the occurrence of early bud virus (BV) and late inclusion body derived Virus (ODV) maturation process.