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International Journal of Virology
Year: 2005  |  Volume: 1  |  Issue: 1  |  Page No.: 6 - 6

Large Scale Production of Potato Virus Y Necrotic Strain (PVYN) Coat Protein Through Expression the cp Gene in E. coli.

A. S., Gamal El din, Sohair, El-Afifi, A. S., Sadik, Nashwa, M.A. Abd El- Mohsen and H. M., Abdelmaksoud    

Abstract: A fragment with a size of 801 bp from the cp gene was amplified from potato virus YN. RNA extracted from virus infected tissues of D. metel leaves by the use of degenerate primers through polymerase chain reaction (PCR). Large-scale amount of PVY (N Egypt) coat protein produced by gene expression technique in E. coli was through: (1) Insertion cp gene isolated by RT-PCR into PinPointTM Xa-1 vector by ligation and propagated by transformation process in E. coli. (2) Isolation of plasmid DNA, then used restriction enzymes BamH I and Bgl II to identify clones containing inserts. To confirm the fragment inserted of cp gene sequence PCR was performed using specific primers for PVYN cp gene. (3) The gene expression was performed using 100 µM IPTG & 2 μM biotin and incubation for 4h/37 C° to produce biotinylated protein for E. coli with Mr 22.5 KDa and fusion protein with Mr ~48 KDa consist of biotin tag and CP product of PVYN. (4) The protein was purified by soft linkTM soft release avidin resin. About 2.85 mg/mI of the expressed protein was purified from 1 L of bacterial culture.

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