Characterization of a homologous-region-binding protein from white spot syndrome virus by phage display.
Homologous regions (hrs) of white spot syndrome virus (WSSV) might serve as origins of DNA replication or be involved in transcriptional regulation. To characterize the interaction between hrs of WSSV and the viral proteins, in this investigation, phage display technology was used. WSSV genomic DNA was sheared by sonication to generate fragments in lengths between 0.5 and 2.0 kb. Then these fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of rebuilt vector pCANTAB 5 EE to obtain WSSV genome phage display library. Using a 210 bp DNA from the b minifragment of WSSV hr2 as the bait, biopanning of WSSV genome phage display library for five rounds resulted in the isolation of a recombinant phage clone containing an exogenous DNA fragment of 306 bp. This DNA fragment was identified to be the 5' terminus of the wsv021 open reading frame in WSSV genome. Temporal transcription analysis revealed that the wsv021 gene was transcribed at the early stage of WSSV infection. The gene was expressed as a fusion protein in Escherichia coli XL1-Blue. The electrophoretic mobility shift assay indicated that the recombinant WSV021 protein (rWSV021) could bind specifically to the 210 bp DNA from the b minifragment of WSSV hr2. The wsv021 gene might be a functional gene involved in WSSV replication and transcriptional regulation.
Zhu
Yanbing
Y
School of Biotechnology, Jimei University, Xiamen 361005, People's Republic of China.
Ding
Qi
Q
Yang
Feng
F
D001483:Base Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*; D046848:White spot syndrome virus 1 / Q000235:genetics*
D004268:DNA-Binding Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins