菜花烙铁头蛇毒去整合素的纯化、克隆及活性研究

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	菜花烙铁头蛇毒去整合素的纯化、克隆及活性研究
其他题名	Studies of purification, cloning and biological activity of disintegrins from Trimeresurus jerdonii venom
	周兴丁
学位类型	博士
导师	熊郁良
	2004-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	菜花烙铁头蛇毒去整合素血小板肿瘤精卵结合
摘要	蛇毒去整合素蛋白（disintegrin）家族一般具有分子量低、富含半胧氨酸和含有RGD三肽等结构特点，因其能与配基竞争结合细胞表面的整合素受体，干扰整合素的正常功能而呈现出多方面的生物学活性，如抑制血小板聚集、抑制肿瘤转移和诱导细胞凋亡等。本论文用凝胶过滤、反向高压液相等层析技术从我国云南产菜花烙铁头（Trimeresurusjerdonii）蛇毒中分离纯化到两个新的去整合素蛋白，分别命名为jerdonin和Jerdonatin。MALDI-TOF-MS测定jerdonin分子量为7483Da，jerdonatin分子量为80llDa，二者均属第二类去整合素。分别测定了其N末端25个氨基酸序列，发现与其它蛇毒来源的去整合素具有很高的相似性。提取菜花烙铁头毒腺总mRNA，通过反转录PCR（RT-PCR）扩增到两个去整合素cDNA序列，分别命名为TJDIS-1和TJDIS-2。TJDIS-1全长1528bp，TJDIS-2全长16O3bp，二者均包含编码信号肚区、前肤区、金属蛋白酶区、间隔肤区和去整合素区一属于P一H型金属蛋白酶家族。经推导的氨基酸序列和MALDI一TOF一MS分析，可确定TJDIS-1去整合素区编码jerdonin，TJDIS-2去整合素区编码jerdonotin。jerdonin全长71个氨基酸，jerdonatin全长72个氨基酸，二者均含12个半眺氨酸，且在C末端附近均含有去整合素家族的典型特征三肤序列一RGD。氨基酸序列比较分析显示，jerdon加与来源于侏响尾蛇（Sistrurusm.tergeminus）的去整合素tergeminin序列相似性最高，为82.2％。而jertionatin与来源于黄绿烙铁头（Trimeresurusflavoviridis）的去整合素CTF-I序列相似性最高，为97.2％，仅在RGD附近有两个氨基酸不同。jerdonin与jerdonatin序列相似性为67.6％。本论文还研究了jedonin和jerdonatin的生物学活性。二者均能强烈抑制ADP、胶原、凝血酶诱导的人血小板的聚集，IC50为100-300nM。jerdonin和jerdonatin还能够抑制B16肿瘤细胞增生，研究表明，jerdonin不仅能显著抑制肿块的生长，还能延长荷瘤小鼠的寿命。平均肿块体积由对照的5260．33枷，降低至2086.65mm3，小鼠平均寿命由对照的24.8天延长至30.5天，提高了22.98％。本论文也研究了jerdonin和jedonatin对小鼠精卵受精过程的影响，结果表明jordonotin能够呈剂量关系抑制精卵结合，但对精卵融合无影响。当jerdonatin用量为10.0μg／毗时，单个卵子的精子结合数由对照的95.31降为21.83。菜花烙铁头蛇毒去整合素蛋白的结构和活性鉴定，为去整合素家族的生物多样性研究提供了丰富的素材，同时也为血栓、肿瘤、生殖等生理或病理过程的研究提供了一个有力的工具。
其他摘要	Disintegrins are a family of low-molecular-weight, cysteine-rich and RGD-containing peptides that bind specifically to integrins expressed on cell surfaces. Disintegrins can act on many biological processes such as platelet aggregation, tumor metastasis, and induction of apoptosis. In this study, two novel disintegrins, jerdonin and jerdonatin, were purified to homogeneity from Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. By matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) molecular mass of jerdonin and jerdonatin were determined to be 7483Da and 801IDa, respectively. Jerdonin and jerdonatin belong to medium weight disintegrin subfamily. 25 N-terminus were sequenced and shared high similarity with other disintegrins from snake venom. In addition, two cDNAs encoding jerdonin (TJDIS-1) and jerdonatin (TJDIS-2) were cloned from venom gland of Trimeresurus jerdonii by reverse transcription polymerase chain reaction (RTHPCR). The full length cDNA of TJDIS-1 is 1528bp, That of TJDIS-2 is 1603bp. They both encode signal peptide, pre-peptide, metalloproteinase and disintegrin domains. From the deduced ami no acids, jerdonin is composed of 71 amino acid residues and jerdonatin is composed of 72 amino acid residues. They both include 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD) near the C-terminus, a well-known characteristic of the disintegrin family. Amino acid sequence of jerdonin is proved to be greater similarity (82.2%) to that of tergeminin from Sistrurus ID. tergeminus, That of jerdostatin has great similarity (97.2%) with CTF-I from Trimeresurus flavoviridis and shares 67.6% similarity with jerdonin. Furthermore, the biological activities were characterized. Jerdonin and jerdonatin potently inhibited ADP-, collagen- and thrombin-induced human platelet aggregation with IC50 of 100-300nM. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice. The mean tumor volume decreased from 5260.33 mm3 to 2086.65 mm3, and the average survival time of the mice improved 22. 98% from 24. 8 days to 30. 5 days. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to the mice zona-free eggs and their subsequent fusion. Jerdostatin significantly inhibited the sperm-egg binding in a concentration-dependent manner but had no effect on the fusion of sperm-egg. When the dose of jerdonatin was 10. 0 jig/ml, the number of sperm binding In each egg fell to 21.83 from 95.31 of the control. The results of the structure and function of disintegrins from Trimeresurus jerdonii are material of studying the biological diversity of disintegrin family, and the disintegrins are probably useful tools to study some physiological and pathological process such as thrombus, tumor, and fertilization.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/6188
专题	其他
推荐引用方式 GB/T 7714	周兴丁. 菜花烙铁头蛇毒去整合素的纯化、克隆及活性研究[D]. 北京. 中国科学院研究生院,2004.

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