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题名	蟾蜍皮肤活性蛋白与多肽的研究
作者	赵宇
学位类别	博士
答辩日期	2005-06
授予单位	中国科学院研究生院
授予地点	北京
导师	张云
关键词	溶菌酶 血红素 丝氨酸蛋白酶抑制剂 cDNA文库 皮肤分泌物 华西蟾蛛
其他题名	Studies of bioactive proteins from Bufo andrewsi skin
中文摘要	近年来，我们致力于蟾蛛（Bufo andrewski）皮肤活性组份的研究，构建了缥蛛皮肤cDNA文库，检测了蟾蛛皮肤分泌液中多种生物活性，进一步纯化得到了四个新的生物活性蛋白：溶菌酶、抗爱滋病毒蛋白以及两个丝氨酸蛋粼酶抑制剂，并，简述如下；I、第三章报导了我们从蟾蛛皮肤分泌液中分离得到的一个谊薇酚犷杰女呱BA一娜"zym"）·BA一lvs"Zym"经5DS一PAGE检测为一条带，其分子量约为巧k珍。·它是一个高效的溶菌酶，每毫克蛋白的溶菌活性为2．7xl护俪ts，还能抑制革兰氏阳性菌（金黄色葡萄球菌Slaj，匆褚ococcusaureus）和革兰氏阴性菌（大肠杆菌Esch邵ichiacoli）生长，其最小抑菌浓度（MIC）分别为1．36拼M和84并M。使用PCR筛选法，我们从蟾蛛皮肤c溯A文库中克隆得到编码BA一lyso即me的cDNA序列。BA一lysozymeN末端测序和肤质量图谱确认了蛋白和基因的网一性。它的蛋白全序列与鸡溶菌酶的相似性为5氏5％。系统发育分析显示，与其最相似的是来源于海龟的溶菌酶。11、第四章介绍了我们从蟋蛛皮肤分泌液中分离得到的一个新的抗lllV蛋白，命名为BAS一AH。BAS一AH是一个分子量为63kD。的单链蛋白，每摩尔蛋白质含有0．89摩尔血红素辅基。BAS一AH对人T淋巴细胞系CS166细胞的毒性（CCS。）为9．5尽M。BAs一AH具有较强的抗HIV活性，它对HIV感染和复制具有剂量依赖抑制效应，其选择指数（CCso／Ecs。）分别为14．4和11．4·BAS一麟J也能抑制HIV的逆转录酶，其1C5（）为L32冬以。BAs一AH的N末端氨基酸为NA以KADvIGKIsILLGQDI』slvAAM，与己知的抗Hlv蛋白没有同源性·表明它可能是一个新的抗川v蛋自。BAs一AH没有检测到抗菌活性、蛋自酶水解活性、胰蛋自酶抑制剂活性、L一氨基酸氧化酶活性和过氧化氢酶活性。班、第五伞报导了我们通过离子交换、分子筛和反向层析，从蟾赊皮肤中分离得到的一个新的胰蛋白酶抑制剂，命名为BATI。BATI是一个单链糖蛋自·其分子量为22kD。。它是吵～个热稳定的竞争性的抑制剂，能有效抑制胰蛋自酶·其抑制常数凡为14nM。B灯I对凝血酶、弹性蛋白酶以及糜蛋白酶都没有抑制作用。BATI的N末端序列为El犯ITD，不同于其它物种来源的蛋白酶抑制剂。W、第六章介绍了蟾蛛皮肤分泌液中纯化得到的另外一个蛋白酶抑制剂（命名为baserpin）。与上述BATI不同的是，basel咖n不可逆地抑制多种蛋白酶。它是一个分子量约为60kDa的单链糖蛋白，除了能抑制胰蛋白酶，还能有效抑制糜蛋白酶和弹性蛋白酶。它抑制上述三种酶的二级反应常数（编）分别为4．6x1护M一，s一l、8．9》1护M一15一I以及6．8xl护M一ls一l。BaserPin是第一个来源于两栖类皮肤的不可逆抑制剂，其N末端氨基酸序列为HTQYPDILIAKPxDK，与其它物种来源的蛋白酶抑制剂不同。本论文综述了蟾蛛皮肤中的活性组份，报导了我们近年来研究蟾蛛皮肤活性蛋白与多肤的进展，分四章详细介绍了蟾蛛皮肤中纯化得到的四个活性蛋白。BA一lysozyme是两栖类动物中第一个得到蛋白质全序列的溶菌酶，能有效抑制革兰氏阳性菌和革兰氏阴性菌生长；BA象AH是一个含血红素辅基的抗HIV蛋白，其独特的理化性质和功能证明它是一个新的抗病毒蛋白。根据所鉴定的性质判断，BATI和bos仰in分别属于竞争性抑制剂和不可逆抑制剂。其中，base印in是第一个从两栖类皮肤中分离得到的不可逆抑制剂。
英文摘要	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.
语种	中文
公开日期	2010-10-15
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6180]
专题	昆明动物研究所_动物活性蛋白多肽组学
推荐引用方式 GB/T 7714	赵宇. 蟾蜍皮肤活性蛋白与多肽的研究[D]. 北京. 中国科学院研究生院. 2005.

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