Authors
Marcia Brady
Summary
Bacillus subtilis is a spore forming, motile, rod-shaped, Gram-positive, facultative aerobe. The entire sequence of its genome is known, which greatly accelerates the application of genetic engineering technology. A variety of eukaryotic and prokaryotic exogenous genes have been cloned and expressed in the Bacillus subtilis expression system. Researchers have successfully constructed the expression vector pMK-BD-2/CP1, and realized the expression of the fusion antimicrobial peptide pBD-2/cecropin P1 in Bacillus subtilis. The expression content is 45 mg/L with validated good antibacterial activity . It can be achieved by artificially synthesizing the target gene sequence of the antimicrobial peptide, linking it with the expression vector pHT43, constructing the recombinant expression plasmids pHT43/SS-BD and pHT43/CC34, transferring them into Bacillus subtilis, and successfully expressing the antibacterial peptides β-defensi and CC31. In addition, the antimicrobial peptide Cathelicidin-BF and Abaecin has also achieved expression in Bacillus subtilis, and the expression contents were 0.5 mg/L and 3 mg/L, respectively. Bacillus subtilis, as a genetically engineered bacteria, provides a good way for the production of antimicrobial peptides.
Introduction
The Bacillus subtilis expression system mainly has the following limitations in the production of antimicrobial peptides:
1) Bacillus subtilis secretes proteases, which will degrade part of the antimicrobial peptides and affect the expression level.
2) The expression efficiency of antibacterial peptides is low, which is also an urgent problem to be solved.
3) The instability of the plasmid is manifested in two aspects, the instability of the structure and the isolation. The former is likely to cause the loss of antimicrobial peptide gene fragments and structural changes, while the latter may lead to the loss of expression plasmids. The use of integrated plasmids is an effective way to solve plasmid instability.
Procedure
Considerable advances have been made in the genetics and molecular biology of lactic acid bacteria, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus spp. These have resulted in the construction of constitutive gene expression cassettes, inducible gene expression systems, and specific protein targeting systems for these bacteria. The modified lactic acid bacteria hardly secrete proteases, which indirectly reduces the risk of antimicrobial peptides being internally decomposed. Therefore, it has some natural advantages compared with other expression systems. Studies have shown that antibacterial peptides have been successfully expressed in lactic acid bacteria, and realized the expression of active antibacterial peptide Baetenecin7 in this system. Humanized antimicrobial peptide LL-37 has also achieved expression in lactic acid bacteria NZ9000. These indicate that the lactic acid bacteria expression system, as a new type of expression system, has the potential to produce antimicrobial peptides. It can also be used as a way for the development and large-scale production of antimicrobial peptides.
The lactic acid bacteria expression system has been gradually known and applied, but there are also some problems and deficiencies. The first is that lactic acid bacteria expression system has few research reports on the production of the antimicrobial peptide. The current research on lactic acid bacteria expression systems is still in the exploratory stage, and the understanding of its genetic background is still very weak, which limits its development. Secondly, low protein expression efficiency and instability of signal peptide secretion are both important factors affecting the production of antimicrobial peptides.
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