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Human Defensin α(HD-α) Gene Fusion Expression, and Its Protein Purification and Bioactivity Detection

Author: ZhangJian
Tutor: ZhaoZhongLiang
School: Fourth Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: human defensin- α recombinat clone chromatographer antimicrobial bioactivity
CLC: Q819
Type: Master's thesis
Year: 2005
Downloads: 63
Quote: 0
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Abstract

Defensins, a group of small canonic antimicrobial peptides with three intramolecular disulphide bonds, were divided into three subfamilies, α-,β- and 8- subfamily. They are all cysteine-rich, cyclic peptides. Defensins distribute widely in human cells and tissues, and play important roles in host defence barrier. They have antimicrobial activities against Gram-positive, Gram-negative bacterium, fungus and virus. α -defensins are secreted most common in neutrophils and Paneth cells (enteric a-defensin) of the small intestine and have many biological effects, such as opsoninzation and assistant effect on macrophage ingesting, binding to the receptor of ACTH and act as antagonists, being chemotactic in vitro for monocytes, and enhancing immuno-response. Recent clinical researchs showed that the changing of defensins in plasma andbody fluids were helpful for diagnosis of some inflammation diseases such as pyohemia, bacterial infection, chronic pulmonary disease and premature labor. Meanwhile, purify or recombination defensins which were regarded as "super antibiotics to replace the traditional antibiotics is becoming hot on pharmacal researchs.The mechanisms of how defensins perform the biological effectives are still not clear. Some evidences show that the positive net charge of denfesins is necessary for binding with bacterium and killing them. Other researches found that the oxygen partial pressure and energy metabolism of tissues influence defensins bioactivity definitely. So, lots of works need to be done. To do the research work on defensin, the basic work is to obatain a mount of purified defensins. For defensin usually locates in some special cells such as macrophage, neutrophil, Paneth cell, it is hard to purify from natural cells. In this work, we try to express the human defensin-a polypeptide by recombination technology, then purify and detect its antimicrobial activity. So as to offer materials for further research.In the study, the defensin sequence was from databases of Gene-Bank, and chemosynthesized. Also a hydroxylamine cleavage site was added in the 5’ end of defensin. The chemosynthesized DNA fragments were then annealed, clonedinto the pBV220-IL4 vector fusioned with IL-4 in order to increase the expression. By transforming the recombinants into competence DH5a and screening with restriction enzymes, finally, a positive clone was obtained and confirmed by sequencing.The recombinant strain was temperature induced to observe the expression of rHD-α fusion protein at 42℃, and to determine the optimized inducing time. Then, the recombinant strain was fermented in fermentor, by method of Dissolve Oxygen Feedback-Nourish Supply in Batch. Harvesting the bacteria by centrifuging, lysis the bacteria by ultrasound, the large munber of inclusion bodies of expression protein were obtained. The inclusion body of expression proteins was then washed by guanidine hydrochloride, purified by Reversed Phase Chromatography, cleavaged by hydroxylamine. Finally, the rHD-α was purified by three types of chromatographer. The renature of rHD-α was proceded by gradually decreasing guanidine hydrochloride concentration with dialysis the protein solution. The final purified proteins were concentrated to 20μ g/ml by ultrafiltrating.To detect the bioactivity of renatured proteins, perforation experiments were performed. A bacteria inhibition zone was observed around the hole where the rHD-a were placed into. This result suggested that the purified rHD-α

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