【作者】 张骞；

【导师】 付学奇；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2004， 硕士

【摘要】 金黄色葡萄球菌(Saphylococcus　aureus,ScA) 属革兰氏阳性球菌，是临床上引起化脓性感染和医院内感染常见的病原菌。其临床表现多种多样，除主要表现为可引起皮肤、粘膜、肺脏等多种组织器官的化脓性炎症外；有些菌株还可引起食物中毒、毒性休克综合征(TSS)、表皮剥脱综合征等严重并发症。在对金黄色葡萄球菌的研究中，人们发现，当将金黄色葡萄球菌在培养基中以最适条件下培养时，其培养物滤液中可生成大量的代谢产物。这些代谢产物成分复杂，包括酶类、毒素、A蛋白等，它们与金黄色葡萄球菌的致病性及耐药性密切相关，属于金黄色葡萄球菌的毒力因子。但但近年来，人们在研究中却发现，这些代谢产物其中不少在一定浓度和不同途径给予时,不仅没有致病作用，却对人与动物的许多疾病有着良好的治疗作用。金黄色葡萄球菌滤液既为根据金黄色葡萄球菌代谢产物的治疗作用研制开发的一类新药，2000年收入中国生物制品规程。其主要是用于治疗骨折，后发现该药对因放化疗而导致的白细胞减少也有一定保护作用，有免疫调节作用，扩大了该药品的应用范围。但由于该产品是微生物发酵滤液，成分十分复杂，对其中很多成分功能研究尚不清楚。因此，对其成分的细致研究，对于该药的进一步研究有着重要意义。本实验即主要是对金黄色葡萄球菌滤液中部分有效组分进行研究。主要包括两部分，分别是对金黄色葡萄球菌滤液中促成骨细胞增殖多胺类活性物质的研究，以及对金黄色葡萄球菌滤液中凝固酶的研究。 <WP=67>首先是对金黄色葡萄球菌滤液中促成骨细胞增殖多胺类活性物质的研究。在以往的研究中，已经有人发现金黄色葡萄球菌滤液中含有一种13kDa左右的物质，名为蛋白A的这种物质有着促骨细胞增殖的作用。但近年的研究表明，在金黄色葡萄球菌滤液中有着促骨细胞增殖的作用的物质并不仅此一种，其中尚有其他物质有着类似功能。而在第一部分研究过程中，通过实验发现在本种金黄色葡萄球菌滤液制剂中，具有促进骨细胞增值作用的有效成分除了起着主要作用的蛋白A外，还有一种小分子活性物质也起着同样作用。通过使用Sephadex G-25柱和 zerolite225阳离子交换树脂柱层析对金黄色葡萄球菌滤液纯化，对这种小分子物质进行了分离提纯，然后，通过使用红外光谱分析等方法对此多胺类活性物质进行的研究，证明了这种物质就是精胺。文献中精胺具有促进细胞增殖的作用的报道也证实了这一点。而对于本种金黄色葡萄球菌滤液中大量精胺的来源，根据实验结果判断，应该是此种金黄色葡萄球菌利用培养基中的精氨酸，通过一系列生化反应后产生的。并推断其流程为：首先，将精氨酸转化成鸟氨酸；之后，再利用鸟氨酸脱氨酶，丙胺转移酶等酶类一步步的将鸟氨酸转化成为腐胺，之后转化为亚精胺，最终生成精胺。而第二部分实验中则是对对金黄色葡萄球菌滤液中凝固酶的研究。作为金黄色葡萄球菌中较早被发现的几种代谢产物之一，凝固酶活性早已被作为金黄色葡萄球菌检测标准之一，但对凝固酶的确切功能尚无深入研究。而经研究，在已被批准为国家新药的<WP=68>金黄色葡萄球菌滤液中，发现此种菌液中产生的凝固酶除共有的凝血作用外，还有着抗原性质，于是，对其进行了分离研究工作。这部分的主要工作包括，首先，采用超滤分级，丙酮分级沉淀和凝胶渗透柱层析从金黄色葡萄球菌滤液中分离纯化得到了高比活性凝固酶；用高效液相色谱分析纯度达到95％以上。之后对此纯化后凝固酶进行进一步研究。首先，用凝胶渗透柱层析对该凝固酶分子量进行了测定，测得分子量为35万左右。测定了氨基酸组成，发现色氨酸和酪氨酸含量很低，也因为这两种氨基酸的缺乏，凝固酶在紫外可见吸收光谱上280nm处无特征吸收，这点在紫外全波长扫描时得到了验证。在氨基酸分析过程中发现凝固酶中含有另一种含氨基物质，因为该物质能与FDNB反应生成衍生物。另从氨基酸组成计算，氨基酸总量占该酶的80％左右，说明该酶可能是个复合物。通过定性测定，发现纯化后的凝固酶有糖类的特征反应，由此判断与凝固酶结合的物质是糖类物质，即凝固酶可能是氨基多糖与蛋白质的复合物，这一点与普通抗原有相似之处。而定量计算总糖含量占凝固酶总量约为18.1 %左右，也与前面氨基酸组成计算数据相符，证实了实验前的推测，现在对该物质的进一步研究正在进行当中。金黄色葡萄球菌分泌的游离凝固酶和肠毒素均属于细菌外毒素，现关于肠毒素的报导结果较多，研究证明其是一种超抗原，对它的检测在药品，食品生产中有重要意义，但关于的报道则较少。而对于凝固酶的报道较少，大多仅作为检验肠毒素的方法之一。而根据本实验结果判断，凝固酶本身也具有抗原性，在实验中能促进T淋巴细胞增殖，从这个实验结果看，其本身也很有可能是一种超抗原，需要进一步研究。而凝固酶具有抗原性的事实，<WP=69>则说明在 “金黄色葡萄球菌滤液”的药效方面凝固酶也可能起着作用。但由于凝固酶的凝血作用，在进入机体后往往会产生不良反应，虽无后遗症，但仍应该对凝固酶活性进行一定控制。更多还原

【Abstract】 Staphylococcus aureus (ScA), one kind of Grans+ bacterium, is a type of common pathogenic coccus that always causes nonspecific infection and nosocomial infection. Some ScA may cause suppurative inflammation of many tissues and organs such as skin, mucous membrane and lung. Others can cause food poisoning, TSS, exofoliation syndrome and etc. Staphylococcus aureus can generate plenty of metabolites including enzyme, toxin and protein A,etc, which are virulence factors and correlate with pathogenicity and tolerance of ScA. Recent studies indicated that these metabolites could cure many human diseases without side effect. For example, Staphylococcus aureus filtrate (SAF) is one kind of new drugs developed according to its therapeutical effect. At first, SAF is used to cure cataclasis. But some time latter, the more effect of this medicine was discovered, for instance, it can prevent edleucocytopenia caused by radiotherapy, and can reinforce people immunity. Now, this medicine is mixture, of which the ingredient is very complicated. So, research on this medicine is very necessary, it is the reason that we change this study as my topic.The work mainly centers about two parts: Part I: Research on one kind of activity polyamines isolated from the SAF.In this part, one kind of activity polyamines was isolated from the SAF. This polyamine has the accelerative effects of the cartilage cell. <WP=71>The isolated steps are as following.Firstly, SAF was separated into big molecular group and small molecular group by sephadex G-25 column. According to some reports, the protein A in the big molecular group has the accelerative effects of the cartilage cell. But cell experiments proved that small molecular group has the accelerative effects of the cartilage cell too. So, we believe that there is some kind of activity polyamine in small molecular group.Secondly, one component was obtained from small molecular group by cation exchange resin column chromatography. After the separation by cation exchange resin column, we gained one kind of polyamines from SAF and analyzed by filter-paper chromatography and further purification.After gaining this polyamine by filter-paper chromatography, we identified what it is through anomi acid analysis, HPLC and IR spectrum. Compared SAF to culture medium by amino acid analysis, the concentration of Arg and Orn has the most obvious change. Results of HPLC proved that the concentration of putrescine and spermine increased obviously.At last, by using the IR spectrum of spermine , spermidine, sample, the pure polyamine is identified spermine. Part II: Research on the coagulase isolated from SAF. In this part, the coagulase in SAF was separated and purified, and its character was researched. The coagulase was obtained by several steps, which were ultra filtrate by using 20kDa film, acetone sediment, etc. Then, it was <WP=72>purified through Sephadex G-200.The purity of coagulase separated by Sephadex G-200 column was analyzed by HPLC, and found its purity is over 97%. Molecular weight of the coagulase was determined by gel filtration on a Sephadex G-200 column calibrated with the molecular weight standards, and the molecular weight of coagulase was 350kDa.The amino acid analysis of the coagulase is analyzed by HPLC and paper chromatography. As the result, the amino acid of the coagulase was found very rich. But in this paper we can see, there is a kind of especial substance in the amino acid analysis chart. Through phenol-vitriol method, result showed that the especial substance is sugar, so the coagulase is a kind of glycoprotein, and the content of sugar in the coagulase is 18.1%.The N-terminal amino acid of the coagulase was identified to be Asp by DNS-Cl method.Circular dichroism (CD) was used to detect its secondary structure with wavelength from 200nm to 300nm and got a positive peak. Comparing it with standard polyLys CD , we could draw a conclusion that the content of α-Helix was 27.3％. At last, we studied the coagulase biology characters.更多还原