The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis / 生物工程学报

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. 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. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.

Expression of the immune inhibitors of human B cell activating factor belonging to the tumor necrosis factor family and evaluation of immune response in vaccinated mice / 中华风湿病学杂志

Objective To prepare T-helper cell(Th)epitnpe-modified human soluble B cell activating factor belonging to the tumor necrosis factor family(BAFF)mutants and evaluate their immune response in vaccinated mice.Methods Recombinant cDNAs were cloned and ligated into the prokaryotie expression vec- tor pQE-80L respectively.The recombinant proteins were induced to express by IPTG in E.coli DH5?and purified with Ni-NTA chromatography.BALB/c mice were immunized with recombinant proteins respectively and the titres of the antibodies that were cross-reactive with BAFF in sera were analyzed by ELISA.Inhibiting ability of the antibodies in sera was analyzed by MTT assay.Results The recombinant proteins were highly expressed in E.coli DH5?.After purification,the purity of recombinant proteins was more than 90%.BALB/c mice immunized with recombinant proteins produced high levels of BAFF-specific antibodies.Cell proliferation assay showed that the sera of immunized mice could inhibit the proliferation-inducing activity of recombinant sBAFF and natural sBAFF.Conclusion The immune inhibitors of human BAFF which can induce polyclonal antibodies that are cross-reactive with BAFF are successfully prepared.These results may provide the basis for further study of their therapeutic effects.