Determination And Analysis Of The Complete Genome Sequences Of BmCPV-SZ And The Assembly Characterization Of The Polyhedrin

BmCPV is one of the pathogens which caused intestinal pus in the midgut ofinfected silkworm larvae, resulting in considerable economic loss in sericulture. Cloningand analysis on the genome could provide the datas for the further study of the genomefunction and pathogenic mechanisms, which play an essential role in prevention of thesilkworm disease.A Bombyx mori cypovirus 1 was isolated in Suzhou from infected silkworm larvaeand tentatively assigned Bombyx mori cypovirus strain Suzhou (BmCPV-SZ). After viruspurification, genome extraction, segment separation, RT-PCR and cloning; the completenucleotide sequences of the genome have been determined. In order to understand theevolution of the virus in the family Reoviridae and the function of the encoded proteins,systematic analysis on the genome and encoded proteins was carried out usingbioinformatics methods.According to the cloning and sequential analysis over the polyhedrin gene S10 ofBmCPV-SZ, we cloned S10 segment into pIZT/V5-His vector, obtained recombinanteukaryotic expression vector pIZT/V5-His-polh, and transfected it into BmN cells bylipofectamine. Sieved by antibiotic Zeocin, the transfected cells expressing the targetpolh was gained. PCR detection confirmed that the polyhedrin has been expressed inBmN cells, meanwhile, the crystal-like polyhedra particles in the cytoplasmic oftransgenic cells could be observed. Then,the transgenic cells were infected by themodified linear BmPAk6, and 96 hours later, the polyhedra in the cells were collected,repeated infection preliminarily revealed that the BmPAK6 could be assembly into thepolyhedra.In addition, late expression factor 3 gene (lef-3) fragments of Pieris rapaegranulovirus was sequenced, and bioinformatics analysis on lef-3 gene and its encodingsequences were conducted by using bio-softs.