Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity.
作者： Li-Na Wang , Bing Yu , Guo-Quan Han , Dai-Wen Chen
DOI: 10.1007/S11033-009-9758-1
关键词: Molecular cloning 、 Gene 、 lac operon 、 Escherichia coli 、 Fusion protein 、 Open reading frame 、 Biology 、 Recombinant DNA 、 Molecular biology 、 Drosophila Protein
摘要: Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, cloning, expression and biological activity detection of Attacin A was carried out present study. The full-length open reading frame (ORF) coding for generated using RT-PCR which takes total RNA extracted from Drosophila as the template. inserted directionally into prokaryotic vector pET-32a (+). resulting recombinant plasmid transformed E. coli Rosetta. SDS-PAGE to detect product induced by IPTG. antimicrobial hemolysis were tested vitro after purification. Agarose gel electrophoresis indicated that complete ORF has been cloned successfully stimulated includes 666 bp encodes 221 AA. encoding mature protein amplified PCR containing A, 570 all. analysis demonstrated fusion expressed approximately 39.2 kDa. Biological activities showed peptide exhibited certain several G- bacteria, well minor porcine red blood cells. In conclusion, successfully. It basis further study Attacin.
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