Cloning Of Human Beta-Defensin 3 And Expression Of Its Fusion Protein

In recent years, for the irrational use of antibiotics produces resistant strains and other reasons, bacterial resistance is more and more serious. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides.Defensins are cationic peptides, isolated from mammals, insets and plants and they serve as effect molecules of innate immunity, providing and efficient defense against infections pathogens. A novel antimicrobial peptide, human β-defensin 3(), was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. In order to study in advanced, we need a lot of hβD-3. In our experiments, the active recombinant fusion protein was expressed in Escherichia coli system. Advanced study could be continued based on our experiments.Objectives: To amplify the cDNA encoding the 45 amino acid containing natural form of human P-defensin 3 and clone it into the prokaryotic expression vector. Then express the recombinant protein in E.coli and purify it in order to get active recombinant hβD-3 fusion protein.Materials and Methods: 1. To clone the cDNA encoding the 45 amino acid containing natural form of hβD-3. Extract the total RNA from human tonsil and amplify the hβD-3 specific cDNA sequence using RT-PCR with two special primers based on hβD-3 amino acid sequence in GenBank . Then the amplified product was analyzed on a 2% agarose gel. The RT-PCR product was inserted into pUC18 and the recombinant plasmid pUC18/hβD-3 was sent tosequence (By Sagon company).2. To construct the prokaryotic expression vector and the control vector. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vector pQE-80L/DHFR/hpD-3 was identified by restriction digestion while we also construct the recombinant control vector pQE80L/DHFR with the same strategy.3. To induce the expression of the fusion protein. The recombinant vectors were transformed into E.coli M15 respectively and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. Analyze the expression by SDS-PAGE with 15% SDS- polyacrylamide gel. Identify the expressed target protein by Western Blotting.4. Isolate and purify the target protein. Prepare 50ml E.coli expressing the interesting proteins and harvest the cells and determinate the solubility of the target protein. Completely solubilize inclusion bodies with 8M urea. Add 1ml of 50% Ni-NTA slurry to 4ml lysate and mix gently and load the mixture into an empty column. Then wash and elute the column with different buffer in order to purify the target fusion protein with the interaction of its 6 X His-tag and Ni-NTA matrices . Renature the purified target fusion protein through multistep dialysis. Then unwater the frozen protein in order to store.5. Check and measure the antimicrobial properties of the recombinant hpD-3 fusion protein.Results: (1) The amplification of the cDNA encoding the 45 ammo acid containing natural form of hpD-3. After RT-PCR with special primers, an about 140 bps fragment could be seen on 2% agarose stained with EB. This fragment was sequenced after inserted into pUC18 . The sequencing result show that the cloned cDNA was 135 bps and consistent with the cDNA sequence encoding the hpD-3 mature peptide reported in GenBank. This cDNAsequence has been accepted by GenBank as hpD-3 cloned from Chinese. The number is AF516673.(2) The construction of the recombinant prorarylic expression vector pQE80L/DHFR/hpD-3 and the control vector pQE80L/DHFR. An about 135 bps fragment could be seen after pQE80L/DHFR/hpD-3 vector was digested by Kpn I +HindIII. An about 550 bps and an about 700 bps fragment could be seen after the vector pQE80L/DHFR/hpD-3 was digested by BamH I +Kpn I and by BamH I +HindIII r...