【作者】 黄林华；

【导师】 闫巧娟； 孙艳； 江正强；

【作者基本信息】 中国农业大学 ， 农产品加工及贮藏工程， 2014， 博士

【摘要】 天然植物来源的生物活性蛋白或通过现代生物技术获得的生物酶制剂因其在食品安全或医药领域中具有非常广阔的应用前景,近年来逐渐受到研究者的关注。本文系统研究了米黑根毛霉(R. miehei)CAU432来源的L-天冬酰胺酶的基因克隆表达、纯化、性质及其在焙烤食品和白血病治疗中的应用。L-天冬酰胺酶和植物凝集素都属于肿瘤治疗药物,因此,本文将实验室之前研究的膜荚黄芪凝集素(AML)和米黑根毛霉重组L-天冬酰胺酶分别作用白血病细胞,并研究了两者共同作用的抗肿瘤效果。本文随后进一步研究了膜荚黄芪凝集素的抗肿瘤活性及其作用机理。论文得出的主要结论如下：(1)首次利用保守区序列和快速末端扩增法成功从米黑根毛霉CAU432中克隆得到一个L-天冬酰胺酶基因RmAsnase。该基因cDNA全长2,156bp,完整阅读框(ORF)为2,049bp,编码682个氨基酸。通过同源性比对分析,RmAsnase编码的氨基酸序列和与霍乱弧菌(Vibrio cholerae)的L-天冬酰胺酶的同源性最高且仅为57%。RmAsnase在E. coli中成功诱导表达,通过Ni-IDA亲和层析一步纯化得到电泳级纯酶。该酶为双亚基蛋白,分子量为135kDa,纯化倍数为2.6倍,回收率为48.8%,比酶活为1984.8U mg-1。重组L-天冬酰胺酶RmAsnase最适反应pH值为7.0,在pH4.0-9.0的范围内保持稳定。RmAsnase最适温度为45℃,在低于45℃时比较稳定。RmAsnase对底物专一性较强,以L-谷氨酰胺为底物时的活性仅为L-天冬酰胺酶活力的1.8%。RmAsnase在添加量为10U g-1面粉时能够使面包和饼干中的丙烯酰胺的含量减少90%左右。RmAsnase能够不同程度地抑制K562、U937和Jurkat细胞的体外增殖。RmAsnase和膜荚黄芪凝集素AML作用以上三株肿瘤细胞具有良好的协同效果,而且这种协同效果呈剂量和时间的依赖性。RmAsnase和AML的协同抗肿瘤效果不仅能够减少两种蛋白药物的加药量,还能加速细胞凋亡的诱导,减少药物作用时间。两种抗肿瘤活性蛋白的协同作用诱导的凋亡为caspase依赖性的凋亡信号途径。(2)AML是从膜荚黄芪的根里分离纯化得到的一种半乳糖特异结合的凝集素。AML能不同程度抑制K562,HeLa及U937三种人类肿瘤细胞的体外增殖,其中尤其对K562细胞系的增殖抑制效果最为明显(IC50=15.8μg mL-1)。通过DAPI细胞核染色,DNA片段化,线粒体膜电位以及PI和Annexin Ⅴ双染等实验方法证实膜荚黄芪凝集素通过诱导细胞凋亡而抑制肿瘤细胞体外增殖。利用caspase广谱抑制剂z-VAD-fmk的介入和蛋白免疫印迹反应(Western bolt)研究细胞内凋亡的信号传导途径,结果表明AML诱导K562细胞发生caspase依赖的线粒体途径的凋亡。通过荧光标记法可以观察到AML能粘附到K562细胞表面,AML特异性结合的半乳糖和乳糖能够明显抑制其抗肿瘤活性以及诱导细胞发生凋亡的能力,说明AML通过结合K562细胞表面的半乳糖残基从而触发细胞内部的内源性的caspase依赖性途径的凋亡。更多还原

【Abstract】 Bioactive proteins derived from natural plants or obtained by modern biological technology are of great potential applied in medicine or food industry and have attracted increasing attentions in recent years. In this thesis, the gene cloning, expression, purification, characterization of L-asparaginase (RmAsnase) from Rhizomucor miehei (R. miehei) CAU432and its application in baking goods and leukemia treatment were studied. It is well known that both of plant lectins and L-asparagines are drugs for the treatment in cancer treatment. Therefore, the anticancer activity of combination with AML and RmAsnase towards leukemia cells was studied. Furthermore, we also studied the antitumor activity and its mechanism of Astragalus membranaceus lectin (AML). The main conclusions of this thesis are as follows:Cloning, expression and characterization of L-asparaginase and its application in baked goodsAn L-asparaginase gene(RmAsnase) was firstly cloned from Rhizomucor miehei CAU432using Rapid Amplification of cDNA End (RACE) assay based on the conserved sequence of L-asparaginase. The full-length cDNA of RmAsnase was2,156bp and a complete open reading frame (ORF) was2,049bp which encoding682amino acids. The deduced amino acid sequence of RmAsnase shares the highest identity of only57%with L-asparaginase from Vibrio cholerae (2OCD). RmAsnase was successfully expressed in E. coli. The enzyme was purified by Ni-IDA agarose column with specific activity of1,984.8Umg-1.The purification fold was2.6and the recovery rate was48.8%. The homodimer enzyme had a molecular weight of135kDa. The recombinant L-asparaginase enzyme was optimally active at pH7.0, remaining stable in the pH range of4.0-9.0. RmAsnase displayed excellent tolerance to high temperature, the optimum temperature is45℃, relatively stable below45℃. RmAsnase showed the highest specificity towards L-asparaginase. The specific activity using L-glutamine as substrate was only1.8%compared to its L-asparaginase activity. RmAsnase (10U g-1flour) caused a clear reduction (about90%) of acrylamide levels in bread and cookies which may help it server as potential additive applied in food industry. RmAsnase displayed significant proliferative inhibition towards K562, U937and Jurkat cell lines in vitro. Synergistic antiproliferative activity of RmAsnase and AML was found against three tumor cell lines tested. The synergistic effect was demonstrated dose-and time-dependent. As positive control, L-asparaginase from Escherichia coli could also enhance the antiproliferation of AML. Furthermore, our results showed that the synergistic antitumor effect of RmAsnase and AML not only allow a decrase in the drug dosage, but also accelerate the apoptosis of K562cells. The co-incubation of these two proteins induces a caspase dependent apoptosis on K562cells.Anticancer activity and its mechanism of Astragalus membranaceus lectinAML is a galactose-specific binding lectin isolated from the root of Astragalus membranaceus membrane. AML showed antiproliferative activity against K562, HeLa and U937cell lines in vitro. K562was the most proliferation inhibited cell lines (IC50=15.8μg mL-1). The DAPI nuclear staining, DNA fragmentation, mitochondrial membrane potential and PI and Annexin V staining assays were used to indicate that the cell proliferative inhibition of AML was via induction of apoptosis in K562cells. Moreover, a caspase dependent mitochondrial pathway of K562cell apoptosis induced by AML was investgated using the intervention of broad-spectrum caspase inhibitor z-VAD-fmk and Western boltting. Fluorescence labeling method visually displayed that the AML could adhere to the surface of K562cells. Galactose or lactose can significantly inhibit AML’s antiproliferative activity and cell apoptosis inducing ability. The apoptosis indued by AML was demonstrated binding to galactose or lactose sites on K562cell surface which may trigger the intracellular caspase dependent singal pathway.更多还原