枯草芽胞杆菌DarA蛋白的鉴定及其原核表达

doi:10.11843/j.issn.0366-6964.2021.05.023

Abstract

Abstract: At present, the addition of antibiotics in animal feed is completely banned in China, thus particular attention has been focused on finding novel antibiotic substitutes in the field. Hence, we aimed to isolate the bacteriocin with antibacterial activity produced by Bacillus subtilis SXAU18, and obtain its antibacterial recombinant protein using prokaryotic expression system. In this study, we isolated the target protein by (NH₄)₂SO₄precipitation, chloroform extraction, ultrafiltration and tested its antibacterial activity by SDS-PAGE analysis and Oxford cup diffusion method. Predicted protein was expressed and purified by prokaryotic expression system and AKTA system. The antibacterial activity of the recombinant protein was determined by the Oxford cup diffusion method. We identified a 15 ku protein containing a unique peptide sequence GSSIFGLAPGK from the antibacterial substance produced by Bacillus subtilis SXAU18, which exerts pronounced antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes. The mass spectrometry and bioinformatics analysis indicated that the 15 ku protein is DarA. Recombinant expression of DarA in prokaryotic expression system and its purification revealed that the DarA was mainly expressed in a soluble form and could be purified to homogeneity, which showed as a single band. The antibacterial test showed that the recombinant DarA has antibacterial activity. The results indicate that DarA from Bacillus subtilis SXAU18 have antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes, and this protein can be obtained through recombinant expression.

Key words: alpaca, Bacillus subtilis, bacteriocin, isolation and purification, prokaryotic expression

CLC Number:

Q939.92

YU Xiuju, SUN Zheng, HAN Xiaotao, LI Yuyu, YU Miao, DONG Changsheng. Identification of DarA Protein of Bacillus subtilis and Its Recombinant Expression Using Prokaryotic Expression System[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1378-1385.

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Identification of DarA Protein of Bacillus subtilis and Its Recombinant Expression Using Prokaryotic Expression System

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