| 其他摘要 | Recently, investigations on the proteins of bufonoid toad skin secretions have been conducted in our lab, and four novel bioactive proteins have been isolated and characterized. They are lysozyme, antiviral protein, and two serine protease inhibitors. So the works contain four major parts, described as follow: In chapter 3, a novel lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme was a single chain protein and the apparent molecular weight was about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme was 2.7x105 units/mg, indicating it is a potent lysozjane. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with MIC of 1.36 and 8.4 jiM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicated that B. andrewsi lysozyme is closely related to that from turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme. In chapter 4, a novel protein, named BAS-AH, was purified and characterized from toad Bufo andrewsi skin. BAS-AH was a single chain protein and the apparent molecular weight was about 63 kDa as judged by SDS-PAGE. BAS-AH was characterized of binding of heme (0.89 mol heme/mol protein) as determined by pyridine haemochrome analysis. Fifty percentage cytotoxic concentration (CC5o) of BAS-AH on C8166 cells was 9.5 uM. However, at concentrations that showed little effect on cell viability, BAS-AH displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 14.4 and 11.4, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. BAS-AH also showed inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase (ICSOr=1.32 uM). The N-terminal sequence of BAS-AH was determined to be NAKXKADVIGKISILLGQDNLSNIVAAM, which exhibited little identity with other known anti-HlV-1 protein. BAS-AH is devoid of antibacterial, proteolytic, trypsin inhibitory activity, L-amino acid oxidase activity and catalase activity. In chapter 5, a novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (Kj) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors. In chapter 6, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by successive ion-exchange and gel-filtration chromatography. Baserpin is a single chain glycoprotein, with apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpins superfaraily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 x lo'lvf1 s"1), bovine chymotrypsin (8.9 x lo^"1 s"1) and porcine elastase (6.8 x lO'M"1 s"1) whereas it showed no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors. In this thesis, we reviewed the bioactive components of bufonoid toad skin and reported our work of screening bioactive protein from toad skin. Four groups of bioactive proteins had been isolated and characterized from skin secretions of Bufo andrewsi. BA-lysozyme is the first report on the isolation and primary structure determination of amphibian lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli. BAS-AH is a novel heme-containing protein. It displayed dose dependent inhibition on HIV-1 infection and replication, and also showed inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase. BATI and baserpin are belong to the two type of protease inhibitor: reversible and irreversible protease inhibitor. Baserpin is the first irreversible serine protease inhibitor isolated from amphibian skin. |
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