Part:BBa_K4248001
LL-37
LL-37
Contribution
The innate immune system is a functional and physiological barrier for the body to resist microbial infection, and antimicrobial peptides are important effector molecules of the innate immune system. At present, about 1500 kinds of antimicrobial peptides have been discovered. Such antimicrobial peptides are widely used in cancer cells, Gram-negative and positive bacteria, fungi, viruses, protozoa and so on. The human body mainly produces two types of antimicrobial peptides: defensin family peptides and cathelicidin family peptides. Although there are many members of the defensin family of peptides, cathelicidin has only one antimicrobial peptide product, LL-37. LL-37 is the C-terminal cleavage product of cathelicidin peptide Hcap-18, which directly kills bacteria, fungi and viruses. LL-37 is expressed widely in human body cells, such as epithelial skin cell，bone marrow cells, and neutrophils. It is responsible for immune defense against bacterial invasion, with study showing its effect of significant reduction in attachment of bacteria and biofilm production We deliberately searched the IGEM biological element library and found that LL-37 was first submitted by the BBa_K1162006 team. On this basis, we carried out further molecular cloning construction and corresponding functional experiments, which can provide further information for other IGEM.
1. Construction of the antimicrobial peptide expression plasmids
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 1.), and the plasmid was successfully constructed. So far, we have successfully obtained five recombinant plasmids, which were respectively on the pET28a(+) vector, which can be used to express antimicrobial peptide proteins.
2. Protein expression and purification
In order to obtain the five antimicrobial peptide proteins, we transferred the recombinant plasmids into E.coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the antibacterial peptide protein we wanted. The concentration of protein was measured as: 0.42mg/mL LL-37. At this point, we got the five antimicrobial peptide protein solutions we wanted.
3. Antibacterial ability test
To confirm the ability of the our purified antimicrobial peptide to inhibit bacterial growth, we used E.coli DH5-alpha as bacteria, and antibiotics as a positive control for bacteriostatic test experiments.
To better show the relationship between the concentration of antimicrobial peptides and the inhibition of bacterial growth, we added 100 μL of DH5α and 100 μL of different concentrations of the antimicrobial peptide to each of the five test tubes. Our five test tubes were filled with the antimicrobial peptide stock solution and diluted 1, 5, 25, 125, and 625 times solution, and repeated three times for each concentration to form the average data graph with error bars.
The results showed that the five antimicrobial peptide proteins had significant antibacterial effects at double dilution concentrations. A single antimicrobial peptide protein can be seen to inhibit the growth of about 60% of bacteria in the range of 5 to 25 times dilution. The antibacterial effect of the Fusion antimicrobial peptide is the best, with about 60% when diluted 625 times. It shows that our antimicrobial peptide products do have a significant antibacterial function, and the antibacterial function of the fusion antimicrobial peptide is the best. A detailed analysis of the antibacterial effect of each antimicrobial peptide is given below.
We also used the same operation to dilute an antimicrobial peptide called LL37. In this histogram, the heights of antimicrobial peptides that have been diluted 625x and 125x with LB medium are almost the same as that of the negative control group, indicating that the sterilization ability of these two samples is practically absent. In contrast, the sample diluted 25 times LB had a significant sterilizing effect but was not as good as the one with five times. The best sterilization effect was the LL37 stock solution, which could sterilize up to 75.74%.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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