Study On Lef-3 Gene, Lef-1 Gene Cluster From Antheraea Pernyi Nucleopolyhedrovirus And Expression Of α-amylase In Silkworm

The gene encoding single-stranded DNA-binding protein(SSB) late expression factor 3 was cloned from Pst I to Xho I fragment of Antheraea pemyi nucleopolyhedrovirus (ApNPV) genome, which is fisrt found in ApNPV. Nucleotide sequence analysis of fragment revealed that lef-3 is a whole open reading frame of 1110 bp codes for 369 amino acids. Compared to the LEF-3 from other baculovirus, it was shown that the homologous on amino acid level was high. LEF-3 has identified as being essential for baculovirus DNA replication. LEF-3 proteins contain a conserved pattern of amino acids similar to this SSB motif in their amino-terminal regions, indicating that this region may be important for LEF-3 function. Upstream the lef-3 there is a sequence encoded 127 amino acids, but it is a part open reading frame. According to analysis of homologous this nucleotide sequence is similar to open reading frame 73 (ORF) of Orgyia pseudotsugata nucleopolyhedrovirus (OpNPV). At 3'end there is no homologous sequence compared to OpNPV and Choristoneura fumiferana nucleopolyhedrovirus (CfNPV), which indicates particularity of ApNPV.We also fisrt found the gene encoding protein of similar to Autographa californica nucleopolyhedrovirus (AcNPV) ORF 13 ORF 12, LEF-1, truncated Ecdysteroid UDP-glucosyltransferase (EGT), BV/ODV-E26, DA 16, which were also cloned from another Pst I to Xho I fragment of ApNPV genome. Compared to the five genes from other baculovirus, they were shown that the homologous on amino acid level was high. Among the five genes: lef-1 is believed to encode LEF-1. LEF-1 has identified as being essential for baculovirus DNA replication and contains three conserved pattern of amino acids, indicating that these region may be important for LEF-1 function and LEF-1 also has the activity of primase. Downstream the lef-1 there is a sequence encoded 79 amino acids named egt, which encodes EGT. Compared to egt from other baculovirus, it is deficient. da26 encodes BV/ODV-E26, which is the structure protein of budded virus, (BV) and occlusion-derived virus(ODV).The α-amylase from the Gram-positive Alicylobacillus acidocaldarius was one kind of thermoacidophilic enzyme. It was expressed by Bombyx mori NPV expression vector fisrt. The nucleotide sequence of the gene amy was 3 906bp, comprising one open reading frame encoding a polypeptide of 1 301 amino acids, the calculated molecular weight of the a-amylase AMY was about 140KD. AMY was expressed in silkworm larva by injection with the recombinant virus. The expressed products showed amylase activity.