Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Prokaryotic Expression and Purification of Human Beta-Defensin-9

Author MaPeng
Tutor LaiZuo
School Nanjing Agricultural College
Course Zoology
Keywords Human β-defensin -6 Prokaryotic expression Separation and Purification Antibacterial activity
Type Master's thesis
Year 2010
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Antimicrobial peptides (AMPs) are important parts of the natural immune system in vivo. They are usually composed by 12~45 amino acids. They mainly exist in cationic form because of their rich in arginine and lysine residues. At present, different types of antimicrobial peptides from various species, including bacteria, fungi, plants, insects, amphibians, fish, birds, mammals and humans, are separated and identified. Most antimicrobial peptides have broad-spectrum antimicrobial activity. They have a killing effect on Gram-positive bacteria, Gram-negative bacteria and fungi at very low concentrations and some of them even in the nmol concentration can completely inhibit bacterial growth. Compared with traditional antibiotics, the biggest advantage of AMPs is that they work rapidly and hardly lead to drug resistance.Defensins are the largest subfamily of antimicrobial peptides family as an important set of endogenous AMPs. Possesing stable molecular structure and multiple biological activities, they are an important component of innate immunity. According to the different spatial structure, defensins can be divided into three categories:α-defensins,β-defensins andθ-defensins. In the field of animal science, known as a wider antibacterial spectrum,β-defensins hardly lead to drug resistance and toxicity. Because of good research value,β-defensins attract widespread attention and has become a research hotspot in recent years.At home and abroad few about the research of humanβ-defensin-6 has been reported. In this paper, recombinant humanβ-defensin-6 (hDefb6) expression plasmid pET-32a (+)-hDefb6 was constructed by means of genetic engineering and hDefb6 was successfully expressed through pET prokaryotic expression system in Escherichia coli BL21 (DE3). Separated and purified by nickel affinity chromatography, formic acid digestion, Sephadex G-50 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) and so forth, pure hDefb6 was obtained. Antibacterial test results showed that: the minimum concentrations of hDefb6 for inhibiting the growth of Gram-negative bacteria Escherichia coli (ATCC 25922) and fungi Candida albicans (ATCC 2002) are 37μg·mL-1 and 50μg·mL-1, respectively. The inhibition to the growth of gram-positive bacterium Staphylococcus aureus (ATCC 2592) was not detected when the concentration of hDefb6 was 200μg·mL-

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