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To better understand the origination and evolution of lef-3 gene from baculovirus genome and determine the relationships between various viruses at molecular level, late expression factor 3 gene (lef-3) fragments of Pieris rapae granulovirus were obtained through conventional PCR method and sequenced after cloned into T-vector. Then, bioinformatics analysis on lef-3 gene and its encoding sequences were conducted by using bio-softs. Four mutations were appeared in the ORF of cloned lef-3 gene, which did not alter the characteristics of amino acids. It was inferred that PiraGV LEF-3 protein contained 399 amino acids, the molecular weight of which was 3.99 kD. Prediction of the LEF-3 advanced structure and homology comparison between other LEF-3 from various baculoviruses showed that the lef-3 gene might encode the single-stranded DNA-binding protein. The result of BLAST revealed that the lef-3 gene only existed in Lepidoptera host for the baculovirus genome, and the evolution analysis illustrated that lef-3 gene could be divided into 3 groups including one granulovirus (GV) group and 2 nucleopolyhedrovirus (NPV) groups. The selection pressure analysis of GV lef-3 gene coding region showed that the majority of lef-3 genes performed negative selection, while the Ka/Ks differed from different lef-3 gene, to some extent, which also performed positive selection. The origination analysis revealed that lef-3 gene of baculovirus might derive from bacteria. The lef-3 gene of PiraGV was cloned successfully and the possible patterns of origination and evolution were speculated through bioinformatics analysis.