Abstract
This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.
Highlights
Taking candida albicans (ATCC10231) as the indicator, the antifungal activity of recombinant Musca domestica antifungal peptide −1 (MAF-1) fusion protein after purification was detected by Micro liquid–bacterial colony notation
Antimicrobial peptides are the key components of the insect immune system
Anti-microbial peptides are widely distributed among different insects and the presence of these functional peptides varies from species to species[18,19,20,21,22,23]
Summary
Analyzed with DNAman software it was shown that the recombinant MAF-1 protein sequence was a 156 N-terminals amino acid sequence of the MAF-1 mature peptide with an added 35 amino acid residue, which contained the His label sequence (Fig. 1c). A clear expression stripe of protein was found near the molecular weight standard between 18.4 kD and 25.0 kD (the sum of MAF-1 mature peptide and His-tag molecular weight). The supernatant of recombinant plasmid transformed bacteria after splitting contained soluble recombinant MAF-1 fusion protein and a lot of recombinant MAF-1 fusion protein expressions were found in the inclusion body (Fig. 2a) Using a His Bind Purification Kit a purified recombinant MAF-1 fusion protein (from body and bacteria fluid supernatant of pET-28a (+ )-MAF-1 recombinant plasmid transformed bacteria) could be completely eluted by an elution buffer solution containing 50 mM 100 mM imidazole (Fig. 2b). Taking candida albicans (ATCC10231) as the indicator, the antifungal activity of recombinant MAF-1 fusion protein after purification was detected by Micro liquid–bacterial colony notation. Compared with negative control group, the colony number of MAF-1 treatment group was significant decreased (Fig. 3)
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