| Most of conventional antibiotics are produced by molds and actinomycetes, and chemically they include β-lactams, macrolides. quinolones, tetracyclines, aminoglycosides, and so on. Antimicrobial peptides are a new class of antibiotics discovered recent years, which are encoded by genes of organisms and chemically peptides, so they are also called as peptide antibiotics. Many organism species, ranging from plants, insects to lower vertebrates and mammals, can produce peptide antibiotics. They contribute to innate host defense against a number of bacterial and fungal pathogens, even some of the pathogens resistant to conventional antibiotics.More than 300 peptide antibiotics have been found so far, and some have natural or artificial mutants. In order to obtain a new peptide antibiotics mutant molecule with strong antimicrobial activity and/or more simple structure, a mutant library was often constructed by genetical method and then the desirable mutant molecule was screened out from the library.In this dissertation, the following experiments are conducted:Firstly, Cloning of hPAB- β gene, i) The amino acids sequences of 23 animal β -peptide antibiotics were analyzed by multiple sequences alignment method and a pair of degenerated primers were designed according to the consensus sequence derived from mature peptides of some beta peptideantibiotics. ii)Two fragments (about 240bp and 130bp) were amplified from human keratinocytes total RNA by RT-PCR. The recombinant pM-hPABL and pM-hPABS plasmids were constructed by inserting 240bp and 130bp PCR products into pMD 18-T vector, respectively. The recombinants were identified by restriction enzyme analysis and DNA sequencing, iii) Two ORFs (>100bp) were found in the insert sequence of pM-hPABL. The second ORF (147bp) might be a pseudogene, which actually is the 3' terminal of the first ORF (192bp). BLASTn search result of ORF 192 shows high similar to animal beta peptide antibiotics, being 41.14%, 76.56%, 55.21% nucleotide identity to human peptide antibiotics hBD-1, hBD-2, h.BD-3, respectively. So the cloned gene (ORF 192) may be a new member of human beta peptide antibiotics family, being termed as human peptide antibiotics- & (hPAB ). iv) Signalp analysis revealed this peptide is consisted of signal peptide (22 a.a.) and mature peptide (41 a.a.), having molecular weight 4333.04Da and isoelectric point 8.791.Secondly, determination of the antimicrobial activities of synthetic hPAB- . i) The mature peptide of hPAB was synthesized by Fomc method and purified by reverse phase HPLC. The molecular weight of synthesis peptide was 4334.96Da characterized by mass spectrometry, highly agreed with the theoretical MW of hPAB- 3 . ii) The renaturation of synthetic hPAB was carried out in glutathione oxidation/ reduction solution(l:10). The renatured synthetic hPAB could kill Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus on LB agar plates.Thirdly, construction of artificial mutants of hPAB. i) Two mutants (hPAB-38 and hPAB-34) were designed based on the three-dimension structure of hPAB- constructed by protein homology modeling method, ii) The mutant molecules were generated by PCR and inserted into pFAST HTa donor plasmid, the later was then transformed into Escherichia coli DHlOBacto generate recombinant baculoviruses DNA by site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) propagated in E. coli. The recombinant bacmids were isolated and transfected into insect Sf-9 cells to reproduce baculoviruses. Unfortunately, the stable infectious baculoviruses were not obtained in this experiment.Forthly, high-level prokaryotic expression of hPAB and its mutants, i) The PCR products of hPAB , hPAB 38, hPAB 34 were inserted into pinpoint Xa-3 expression plasmids and transformed into E. coli JM109. The recombinant pinpoint/hPAB expressed unstably in JM109, the interest fusion protein bands being invisible in SDS-PAGE electrophoresis after 5 passages, ii) Pinpoint/hPAB transformed E. coli B...
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