【作者】 吴传芳；

【导师】 鲍锦库；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本文对石蒜凝集素的研究主要包括纯化、性质、活性以及结构几个方面。石蒜（Lycoris radiaca）球茎经匀浆、浸取、硫酸铵分级沉淀得到粗品，将粗品过阳离子交换柱（CM-Sepharose）、阴离子交换柱（DEAE-Sepharose）和SephacrylS-100分子筛层析得到凝集素纯品。经SDS-PAGE检测为单一蛋白带，亚基分子量为12KD，SephacrylS-100凝胶过滤测得其表观分子量为45KD，表明LRA是由四个相同亚基组成的蛋白。LRA能凝集兔血细胞和大肠杆菌，其凝集兔血细胞和大肠杆菌细胞的效价分别为0.95 ug／mL和1.9ug／mL，对外周血淋巴细胞具有很强的促有丝分裂能力；糖抑制实验结果表明，甘露聚糖和甲状腺球蛋白能抑制LRA的凝血活性；对酵母细胞的凝集实验表明，LRA能凝集啤酒酵母细胞；在抗病毒活性方面，LRA能有效地降低Ⅱ型人类单纯疱疹病毒（HSV-Ⅱ）对Vero细胞（非洲绿猴肾细胞）的感染，在500ug／mL时对正常Vero细胞无细胞毒性，IC₅₀=5～10ug／mL之间；在抗爱滋病病毒实验中，LRA能有效降低HIV-1和HIV-2对HT-4和CEM细胞的感染，LRA对HIV-1的半抑制浓度EC₅₀分别为0.43ug／mL和0.79ug／mL，对HIV-2的EC₅₀分别为0.60ug／mL和0.59ug／mL，而LRA对HT-4和CEM的半数细胞毒性浓度为71ug／mL，表明LRA是一种有效的抗病毒蛋白。 LRA的远紫外圆二色谱显示222nm处的单一负峰，208nm和238nm处的肩，此时的LRA分子是一种特异性的高β－折叠蛋白质。LRA的荧光光谱研究表明在激发光波长为280nm时，其最大荧光发射峰在338nm处，荧光光谱未见有酪氨酸（Tyr）残基的发射峰，表明Tyr残基的荧光基本上通过能量转移到TrP上，使荧光强度增强，在激发光谱为295nm时，其最大荧光发射峰338nm，比游离TrP的最大荧光发射峰（348lun）蓝移了近10nln，说明TrP周围的极性较弱，处于疏水的微环境。研究不同温度、PH和基团特异性化学修饰后LRA凝血活性和促淋巴细胞有丝分裂的变化、圆二色谱和荧光光谱的变化，当温度达80℃以上时，活性开始下降，到100℃时活性有60%保留:当pH为2时，活性保留50%，pH为4一12对活性的影响不大;用NBS修饰TrP后，T即的旦一叫睬基的破坏使活性完全丧失，表明TrP对凝血活性是至关重要的，Arg、Tyr、Glu、Asp被修饰后，LRA的凝血活性并未受到大的影响，但Tyr修饰后LRA的促有丝分裂活性降低.用DEPC对组氨酸的p一咪哇基进行特异化学修饰后，其凝血活性有明显的下降，表明p一咪哇基是凝血活性的必需基团。而圆二色谱的研究表明结构的变化先于活性的变化，先是结构开始变化，但活性中心结构并未破坏，接着结构的进一步变化使活性逐步丧失，表明LRA具有较强的抗变性能力。研究了不同温度、pH和特异化学修饰后LRA的荧光光谱变化，发现温度的升高和PH的改变对荧光光谱影响不大，Tyr的修饰使荧光强度降低;丙烯酞胺、KI和氯化艳均能够使LRA的荧光淬灭，丙烯酞胺能淬灭93.6%色氨酸残基的荧光:用NEM做修饰剂测定LRA的可反应疏基数和总琉基数，发现LRA中不含琉基，用比色法测定LRA中TrP的含量为5.3%，用NBS修饰时，测得每分子LRA中含有12.4个TrP残基，即每个亚基约含3个TrP残基。更多还原

【Abstract】 The study on Lycoris radiata Agglutinin (IRA) main include the purification and characterization of LRA, the relationship between structure and bioactivity of LRA. LRA was isolated from the bulbs of Lycoris radiata by extraction, precipitation with (NHJiSO:,, ion-exchange chromatography on CM-Sepharose, DEAE- Sepharose and followed by gel filtration on Sephacryl S-100. The purified lectin showed a single protein band on SDS-PAGE, and the subunit molecular weight was 12KD. The molecular weight was 45KD determined by gel filtration on Sephacryl S-100. The result implied that there are four same subunits per LRA. LRA can agglutinate rabbit erythrocyte at 0.95ug/mL and E. coli cell at 1.9 ug/mL, and also can agglutinate Sacch. cerevisiae cells. The result of carbohydrate inhibition assay showed that Mannan and Thyroglobulin can inhibit the agglutinating activity of LRA. The LRA has strong mitogenetic activity toward T-lymphocyte. LRA showed antivirus activity to the Herpes simplex virus II (HSV-II), and the IC50=5~10 ug/mL, and it showed no cytotoxic activity toward vero cell proliferation at 500ug/mL; In the trials of inhibit HIV-1 and HIV-2, LRA could inhibit the infection of the HT-4 cell and CEM cell by the human immunodeficiency virus-1 (HIV-1) and HIV-2, theEC50=0. 43 ug/mL and 0. 79 ug/mL on inhibit the HIV-I, and the EC50=0. 60 ug/mL and 0. 59 ug/mL on inhibit the HIV-2. It showed no cytotoxic activity toward HT-4 and CEM cell proliferation at 71ug/mL. So LRA is a potent antivirus protein.The LRA exhibited a characteristic circular dichroic (CD) spectrum, a negative band centered at 222nm. It was estimated that LRA was a high - sheet protein. The fluorescence spectrum (FLS) of LRA excited at 280nm and 295nm showed a maximum peak at 338nm. The characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp. When LRA was excited at 295nm, the FLS showed a maximum peak at 338nm, the max of fluorescence emission spectrum blue-shifted more than 10nm compared with the max of free Tyr (348nm).The change of agglutinating activity , CD spectrum and FLS of LRA in different temperature, pH and different chemicals indicated that LRA had partial hemagglutinating activity at pH2. 0(50%), a temperature above 100 (60%) and after modified by N-bromosuccinimide (MBS), the activity lost completely , modified by DEPC, the LRA had a little activity, the other groups modified such as Arg, Tyr, Glu, Asp didn’t effect the hemagglutinating activity of LRA. The result indicated that Trp residues were essential to the hemagglutinating activity and were involved in carbohydrate-binding site.The CD spectrum showed that the denaturation was a two-steps’ progress. First, LRA didn’t lose activity despite of its structure change because the change was out of the active center. Second, the active center was destroyed and LRA began to lose its agglutinate activity. So LRA has a comparatively stable structure. The FLS of LRA with different temperature and pH also showed there was little change. The study on fluorescence quenching showed that the fluorescence from Trpresidues were quenched by KI, acrylamide and CsCl, and 93. 6% of Trp were quenched by acrylamide. No sulfhydryl group has been detected when using N-ethymaleimide (MEM) as a modificatory reagent. There were 5. 3% Trp residues in a LRA molecular determined by colorimetry. Using NBS as the modificatory reagent, there were 12. 4 Trp residues in a LRA molecule. It means that every subunit contains three Trp residues.更多还原