Research On The Protein Interaction Between TRBIV MCP And Spleen Proteins Of Turbot, Scophthalmus Maximus

Turbot reddish body iridovirus(TRBIV) is the causative agent of viral reddish body syndrome(VRBS) that afflicts farmed turbot. Since 2001,VRBS has caused severe mortalities of farmed turbot and led to serious economic losses in China.The major capsid protein(MCP) plays an important role in intrusion and infection of TRBIV. Yeast two-hybrid system has been utilized in this research for screening of the interaction proteins of TRBIV MCP.The pENTR Directional TOPO Cloning Kit has been used to construct the pENTR-MCP entry clone.This technology utilizes a highly efficient, 5-minute cloning strategy to directionally clone the blunt-end MCP PCR product into the pENTR/D-TOPO vector for entry into the Gateway System,and then generate the expression construct pDEST32-MCP by performing an LR recombination reaction. Identify insert-containing plasmid by restriction and sequencing.The author has constructed a cDNA library of turbot spleen cells using CloneMiner cDNA Library Construction Kit. A highquality cDNA library has been constructed without the use of traditional restriction enzyme cloning methods. mRNA has been isolated from total RNA by Oligotex mRNA Kits.Single-stranded mRNA has been converted into double stranded cDNA containing attB sequences on each end.Through site-specific recombination, attB-flanked cDNA is cloned directly into the pDONR222 vector which contains an attP sequences without the use of restriction digestion or ligation. According to calculating, the titer of the amplified library is 2.52×107 cfu/ml, most of the inserts are between 500-1,000 bp, with an average size of about 787.6 bp.pDEST32-MCP has been transformed into the yeast strain MaV203 and its self -activations has been tested by selective plate with 3AT. pDEST22-cDNA has been generated by performing an LR recombination reaction between pDONR222-cDNA and the Gateway destination vector pDEST22.Identifying positive clones in selective plates. By sequencing, 3 cDNA fragments named p22,p23 and p31, have been screened. The size of cDNA inserts are 1117bp ,891bp and 491bp,and p31 is a part of p23. By comparing with GeneBank Database using the BLAST program, p23 fragment has significant homologous with Ribosomal Protein L23,while p22 has no significant homologous sequence.The other 4 clones have been identified not belong to the pDEST22-cDNA library.