Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification

doi:10.13560/j.cnki.biotech.bull.1985.2019-0157

Abstract

Abstract: By analyzing the transcriptome data of Amiota okadai,an attacin homologous unigene named aoattacin was obtained,which was expressed in insect cell Sf9 and its antibacterial activity was detected. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis,and the sequence of the whole gene was synthesized to construct the recombinant pFast-bac1-aoattacin vector. The recombinant vector was transfected into DH10Bac cells to obtain recombinant baculovirus,which was transfected into insect Sf9 cells. To obtain the P1 virus and obtain the P2 virus by amplification,and detect the expression of the gene in Sf9 cells. Protein expression was detected by SDS-PAGE and Western Blot,and the antibacterial activity of the expressed product was determined by the minimum inhibitory concentration. The results showed that a high-expressed aoattacin gene screening from transcriptome data was successfully expressed in insect cell Sf9,and Aoattacin demonstrated antibacterial activity against many kinds of bacteria in experiments,such as both Gram-positive bacteria and Gram-negative bacteria,which may be a new broad-spectrum antimicrobial peptide.

Key words: Amiota okadai, transcriptome, antimicrobial peptide, Eukaryotic expression, antibacterial activity

CAI Juan, LIU Liu, WANG Ling-jun, CAO Jian-ping, ZHENG Ming-hui, LIU Hui. Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification[J]. Biotechnology Bulletin, 2019, 35(9): 118-124.

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