tailieunhanh - Cloning, expression and in silico characterization of a truncated antiviral protein gene from Bougainvillea spectabilis willd.

Bougainvillea antiviral protein (BAP) is one among a class of the ribosomal inactivating proteins isolated from Bougainvillea spectabilis willd. Truncated version of the BAP gene was cloned and expressed in a prokaryotic vector to abolish its cytotoxicity. RNA was isolated from mature leaves of Bougainvillea and the full length cDNA was amplified by reverse transcription-PCR using template mRNA. This full length cDNA of size 756 bp was amplified using the proofreading polymerase (Q5 polymerase) and end to end gene specific primers for removal of C-terminal, the amplicon was cloned in vector by blunt end cloning method. Restriction digestion was performed to release the fragment which was further ligated into prokaryotic expression vector pET29a. The recombinant plasmid was transferred into expression strain BL21 (DE3) and the truncated-BAP gene was expressed by isopropyl β-D thiogalactopyranoside (IPTG) induction. Transformed colonies expressed recombinant fusion Bougainvillea antiviral protein of molecular weight ~ kDa, the size expected for the truncated BAP gene. | Cloning, expression and in silico characterization of a truncated antiviral protein gene from Bougainvillea spectabilis willd.