Characterization Of SpltMNPV Ⅱ ORF59 Gene

The family Baculoviridae is a group of enveloped double-stranded DNA viruses that are highly selective pathogens in arthropods,mainly in insects of the order Lepidoptera.Baculoviruses have been extensively studied and developed as alternatives to chemical insecticides in agriculture and forestry,in both natural and engineered forms,because they possess several properties as biological pest control agents,including higher efficacy in controlling insect pests and a less negative impact on the environment and nontarget species than the chemical pesticides.The baculovirus expression vector system,recombinant baculovirus as bioinsecticide,baculovirus as gene transfer vector and baculovirus surface display have been developed to explore its usefulness for human being. Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) is a newly found virus strain which has a high rate of production and much stronger infectivity than that of SpltMNPV I (Chinese strain).It was first isolated in Japan by Kamiya etc. in the year of 2004. The host of this virus is a kind of polyphagous insect which is harmful to economic crop and vegetables such as alimentary crop,cotton,oil plants and so on. And it is also one of the primary insects pests around the world. Present, SpltMNPV II is considered as the most effective insecticide against the insect, therefore, the researches on it are becoming more and more popular. The whole length sequence of the virus was determined, in which 149 open reading frames were analyzed. Some of the genes of SpltMNPV II have been determined and most of them remain unknown for their functions.The function of SpltMNPV II orf59 gene and its homologues has not been reported so far. The ORF59 was found to be one of ODV proteins by the mass spectrum.This paper presents the characterization of ORF59. The main conclusions are as follows.1. The orf59 gene was cloned from SpltMNPV II genome.It is 645bp long ,potentially encoding a protein of 214 amino acids(aa) with a predicted molecular weight of 25.4kD. This nuclotide sequence has been submitted to the NCBI GenBank and the accession number is NC₀11616. Protein's second structure was analyzed based on the sequence of amino acids.2. By costructing E. coli expression vector, orf59 gene was highly expressed with the induction of IPTG,and its expression quantity reached the highest at the fourth hour,then identified by Western Blotting.The expressed protein in E.coli was purified and used as an immunogen to raise ORF59-specific antiserum in male rabbits.And we find that this antiserum is efficient enough to be used in the following experiments.3. We analyzed the transcription time course of orf59 gene and its localization in BV and ODV,as well as in cells. RT-PCR and Realtime PCR results showed transcript of approximate 645bp was detected at 3 h p.i. and remain detectabe until 72 h p.i.,suggesting that ORF59 protein was synthesized both in early and late course.This is consistent with the sequence analysis result.By the detection of western blotting using polyclonal antibody, a specific immunoreactive band of approximately 29kD was detected in ODV,without association with BV .This was in tune with the analysis of mass spectrum.4.The recombinant virus marked by egfp was constructed, orf59 was inactive. The fragment of pph-egfp was inserted into this vector between the 5'end and 3'end-flanking fragments of SpltMNPV II orf59 gene tandem linked into pUC19. The spli cells were cotransfected with pâ–³orf59-egfp and the wild SpltMNPV II genome DNA. The recombinant virus containing egfp was selected.