藻蓝蛋白α亚基裂合酶的分子克隆和酶动力学研究

为了比较研究不同藻种中藻蓝蛋白裂合酶CpcE/F的结构与功能的差异,对Anabaena sp.PCC 7120中的CpcE/F进行克隆,并进行大量表达,将表达的裂合酶CpcE/F用于藻蓝胆素(PCB)与Mastigocladus laminosus PCC 7603藻蓝蛋白α-亚基(α-PC)脱辅基蛋白(CpcA)的体外重组,得到天然活性的α-PC,从而表明CpcE/F所编码的蛋白质是α-PC生物合成的裂合酶,并对CpcE/F的酶动力学进行了初步研究。     

钝顶螺旋藻藻蓝蛋白的提取及分子量研究

以藻蓝蛋白(CPC)的提取率为指标,用冻融法对藻蓝蛋白进行提取,最后用SDS-PAGE和排阻色谱法对藻蓝蛋白的分子量分别进行了测定,结果表明,冻融法提取藻蓝蛋白的最优工艺参数为:料液比为1∶22,提取温度35℃,提取时间2.5h,藻蓝蛋白的提取率为3.19%。SDS-PAGE法测得藻蓝蛋白的分子量为40000 Da,排阻色谱法测得藻蓝蛋白的分子量为43200 Da,这2种方法所得的CPC蛋白分子量基本一致。     

Se-containing spirulina phycocyanin attenuated liver injury induced by carbon tetrachloride in mice

AIM:To investigate the effect of Se-containing spirulina phycocyanin (Se-SPC) on liver injury of mice induced by carbon tetrachloride (CCl₄). METHODS:The mouse model was conducted by intragastric feeding with 2% CCl₄ oil for three times, meanwhile Se-SPC, spirulina phycocyanin (SPC) and Na₂SeO₃ were injected (ip) to various groups for 7 days. Then selenium (Se), glutathione peroxidase (GPx), superoxide dismutase (SOD), alanine aminotransferase (ALT), malondiaoldehyde (MDA) and nitric oxide (NO) levels in blood and liver were measured. RESULTS:The level of Se,GPx and SOD activities were obviously higher(P<0.05)but ALT activity,MDA and NO₂^-/NO₃^- levels were remarkably lower(P<0.05)in Se-SPC treated groups than those in CCl₄ groups,and effects of high dose Se-SPC on Se,GPx,MDA and NO₂^-/NO₃^- were even more significant(P<0.01).Under the same dose of Se or protein,effects of all selected targets in Se-SPC groups were more efficient than those in SPC groups and inorganic-Se groups.Furthermore,Se levels had a positive correlation with GPx activity(r=01705),which had negative correlation with levels of MDA,NO₂^-/NO₃^- and ALT(r=-0.629,r=-0.336,r=-0.457,respectively), and positive correlations between ALT activity and MDA or NO₂^-/NO₃^- level were found (r=0.519,r=0.641). CONCLUSION:These results indicated that Se-SPC may attenuate liver injury of mice induced by CCl₄ through its anti-inflammatory action and enhancing selenoenzyme expression.     

鱼腥藻藻蓝蛋白的提取

对鱼腥藻藻蓝蛋白的大规模提取方法进行了初步的探索。对实验室精细的小规模分离提取方法进行了简化。用0．01mol／L磷酸盐缓冲液浸提，经3次反复冰融渗透震荡处理，使细胞破碎，释放藻蓝蛋白，然后离心分离。用磷酸缓冲液反复提取至上清液透明。采用这种方法从鱼腥藻中提取藻蓝蛋白，可处理的样品规模大，藻蓝蛋白得率高达98．8％，并且操作方法简便，节约药品，成本低。     

不同冻融条件对破壁提取巢湖水华新鲜蓝藻中藻蓝蛋白的影响

[目的]研究不同冻融条件对破壁提取巢湖水华新鲜蓝藻中藻蓝蛋白的影响,为后续提取纯化试剂级藻蓝蛋白提供支持。[方法]在各种条件下,冻融破壁后,利用粗提液中藻蓝蛋白即PC纯度和得率为评价指标,研究不同冻融次数、不同含水率和不同冻融介质对藻蓝蛋白提取效果的影响。[结果]粗提液中PC纯度随着冻融次数的增加而依次减小,PC得率随着冻融次数的增加呈现下降的趋势;PC纯度和得率随着含水量的增大呈现先持平后降低的趋势;在不同冻融介质的试验中,通过比较得出最优的冻融介质为浓度0．0025mol／L的PBS缓冲溶液。[结论]对于冻融储存时间较长的巢湖新鲜蓝藻,最优的试验条件是选用浓度为0．0025mol／L的PBS缓冲液作为冻融介质,在含水率为96．O％～97．5％区间冻融次数仅需1次,即可得到最优的PC的纯度和得率。     

Effects of phycocyanin on apoptosis of human laryngeal cancer HEP-2 cells

AIM: To investigate the effect of phycocyanin on the apoptosis of human laryngeal cancer HEP-2 cells and to explore the inhibitory mechanism of phycocyanin to tumor. METHODS: Highly purified phycocyanin was extracted from spirulina. The effects of phycocyanin at different concentrations on the growth of human laryngeal cancer HEP-2 cells were detected by MTT assay. In addition, the cell structures were observed under electron microscope. The cell apoptosis was analyzed by flow cytometry. The production of reactive oxygen species (ROS) was measured by flow cytometry. Enzymatic activities of caspase-3, -8 and -9 were measured by chemical colorimatry. The expression of Bax, Bcl-2, Fas, P53, caspase-3 and caspase-9 at mRNA and protein levels was determined by RT-PCR and Western blot. RESULTS: MTT test confirmed that phycocyanin inhibited the cell activity of HEP-2 cells with time and dose dependent manners. The result of electron microscope observation and flow cytometry indicated that phycocyanin induced the apoptosis of HEP-2 cells. The intracellular content of ROS was increased. The activities of caspase-3, -8 and -9 were increased. RT-PCR showed that the mRNA expression of Bax, Fas, P53, caspase-3, caspase-9 was increased and Bcl-2 was decreased. The results of Western blot were consistent with the results of RT-PCR. CONCLUSION: Phycocyanin might induce apoptosis of HEP-2 cells by down-regulating Bcl-2, up-regulating Bax, Fas and P53, and the transduction of apoptotic signals in the human laryngeal cancer cells.     

螺旋藻藻蓝蛋白在水和磷酸缓冲液中稳定性的对比研究

[目的]为了寻找一种代替磷酸缓冲液的提取溶液,满足尽量保持其活性的要求。[方法]分别用蒸馏水和磷酸缓冲液,采用研磨法提取藻蓝蛋白并保存。[结果]两者提取效果差别不大。但是,水保存的藻蓝蛋白溶液比磷酸缓冲液保存的藻蓝蛋白溶液易分解。[结论]综合试验条件及藻蓝蛋白稳定性影响因素考虑,可确定pH变化为藻蓝蛋白分解的主要原因;蒸馏水可代替磷酸缓冲液用于提取藻蓝蛋白,活性维持时间不超过15d。     

鱼腥藻藻蓝蛋白的提取·分离纯化和抑菌研究

[目的]对鱼腥藻藻蓝蛋白的提取进行优化,并对藻蓝蛋白粗品进行分离纯化、凝胶电泳以及抑菌作用研究。[方法]采用反复冻融法提取藻蓝蛋白,液相色谱分离纯化藻蓝蛋白,SDS-PAGE电泳分析蛋白质成分,并利用96孔板法培养、酶标仪浓度检测,进行抑菌作用研究。[结果]藻蓝蛋白提取的最佳条件为固液比1∶1 g/ml,冷冻时间60 min,硫酸铵饱和度为60%。藻蓝蛋白的最大吸收光是620nm。高效液相色谱分离得到3种蛋白质,标记为PC-1、PC-2、PC-3。SDS-PAGE电泳可知,PC-1和PC-2的分子量在14～18 kD。PC-2对苏云金芽孢杆菌和藤黄叠球菌有较弱的抑菌作用。[结论]为鱼腥藻藻蓝蛋白的提取、纯化和性质研究提供了试验依据。     

普通念珠藻(Nostoc commune Vauch.)藻蓝蛋白的提取

[目的]为普通念珠藻藻蓝蛋白的提取及开发提供试验及理论基础。[方法]以普通念珠藻为材料,比较藻体及细胞的破碎方法、提取液类型及饱和硫酸铵浓度对藻蓝蛋白提取的影响。[结果]结果表明:利用发酵法破碎藻体和细胞,0.05 mol/L的KP缓冲液(pH值7.2)作为提取液,经过30%~50%饱和硫酸铵盐析和DEAE-Toyopeal 650 S离子交换柱层析后,藻蓝蛋白纯度达2.51,最大紫外-可见吸收峰位于616 nm。[结论]普通念珠藻藻蓝蛋白分离提取较为理想的程序为:藻粉→0.05 mol/L KP缓冲液(pH值7.2)浸泡→发酵法破碎细胞→35%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S柱层析→较纯的藻蓝蛋白。