天然抗冻多肽的制备、 分离及细菌低温保护活性研究

利用热水抽提法提取鲨鱼皮明胶,控制酶法水解条件制备天然抗冻多肽.以细菌低温保护活性为指标,筛选蛋白水解酶种类及酶解时间,利用Sephadex G-50凝胶过滤色谱、SP-Sephadex C-25强阳离子交换色谱等分离手段进行分离,得到细菌低温保护高活性组分.结果表明,酶解鲨鱼皮明胶得到高活性抗冻多肽的最适蛋白水解酶为酸性蛋白酶,酶解温度50℃、pH 3.0、酶/底物3 000 U·g-1、底物浓度3%、酶解时间1 h;经过Sephadex G-50凝胶过滤色谱、SP-Sephadex C-25强阳离子交换色谱等分离手段得到阳离子的P2组分,经过细菌低温保护测试,当浓度为500μg·mL-1时,大肠杆菌的存活率达到80.8%.分离得到的抗冻多肽具有较高的抗冻活性,可望应用于食品、医药等产业中.     

Analysis of biomarkers for the cross-linkage of formaldehyde with bovine serum albumin peptides

Formaldehyde, a well-known environmental toxic hazard, has been found to produce endogenously via semicarbazide-sensitive amine oxidase-catalyzed oxidative deamination of methylamine. In diabetes, the activity of SSAO has been found to increase with a subsequent increase in endogenous formaldehyde production. It has been postulated that SSAO-induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders. Formaldehyde has also been found to react （cross-link） with amino group of the N-terminal amino acid residue and with the side-chains of arginine, cysteine, histidine and lysine residues. Therefore, formaldehyde may be responsible, at least in part, for protein cross-linkage, oxidative stress and cytotoxicity. The cross-linking of formaldehyde with bovine serum albumin was studied using LC-MS and Mascot database. The peptides sequence for control BSA （untreated） digested with trypsin was matched in the online database search query by exporting the MS/MS data to online MASCOT database. In this way, a total of twenty-seven peptides were matched in the database search query. These twenty-seven peptides were then searched manually in all of the tryptic BSA samples treated with different concentrations of FA that were incubated in different time intervals. Six formaldehyde-treated BSA peptides （FKDLGEEHFK, HLVDEPQNLIK, KVPQVSTPTLVEVSR, RPCFSALTPDETYVPK, LVNELTEFAK, DAFLGSFLYEYSR） were found to be the possible markers for formaldehyde-protein/peptides adducts.     

两栖动物皮肤抗菌肽的研究进展

两栖动物的皮肤抗菌肽是抵御外界微生物侵袭的有效屏障之一.此文综述了两栖动物皮肤抗菌肽的最新研究进展,重点介绍了天然抗菌肽的分离、纯化、基因克隆与表达的过程,并展望了其在农业和临床上的应用前景.     

R9-FOXM1(1-234aa)重组蛋白批量纯化及其抑瘤效应研究

通过基因工程构建重组表达质粒pET15b-R9-FOXM1(1-234aa),转化大肠杆菌建立构建表达R9-FOXM1(1-234aa)的菌株.采用原核表达系统和His-tag亲和纯化手段,规模化制备纯化穿膜肽R9-FOXM1(1-234aa),获得的蛋白的纯度达到90%以上.用R9-FOXM1(1-234aa)穿膜肽处理不同的肿瘤细胞,通过MTT实验研究其细胞效应.结果显示:当R9-FOXM1(1-234aa)穿膜肽的浓度达到2mM时,肿瘤细胞的死亡率为50%左右.实验表明穿膜肽R9-FOXM1(1-234aa)抑制不同肿瘤细胞的生长,有可能成为治疗肿瘤的潜在蛋白类药物.     

Endogenously generated amyloid β increases membrane fluidity in neural 2a cells

The effect of endogenously generated amyloid β on membrane fluidity was investigated in Neural 2a cells stably expressing Swedish mutant amyloid precursor protein (APPswe). Membrane fluidity was studied by fluorescence polarizability using 1,6-Diphenyl-1,3,5-Hexatriene (DPH) as the fluorescence probe. It was found that the membrane fluidity in APPswe cells was significantly higher than that in its wild type counterparts. Alleviating the effect of amyloid β either by γ secretase activity inhibition or by amy...     

Anti-amyloidogenic therapies: strategies for prevention and treatment of Alzheimer’s disease

Deposition of amyloid β-protein (Aβ) in the brain is an early and invariant neuropathological feature of Alzheimer’s disease (AD). The current search for anti-AD drugs is mainly focused on modification of the process of accumulation of Aβ in the brain. Here, we review four anti-amyloidogenic strategies: (i) reduction of Aβ production, which has mainly been approached with secretase inhibition, (ii) promotion of the Aβ degrading catabolic pathway, including an Aβ degrading enzyme, neprilysin, (iii) immunotherapy for Aβ and (iv) inhibition of Aβ aggregation. We have reported that AD patients have a favorable molecular environment for Aβ aggregation and that various compounds, such as polyphenols, interfere with Aβ aggregation and destabilize preformed Aβ fibrils. Received 21 December 2005; received after revision 14 February 2006; accepted 29 March 2006     

超声波在酶法生产紫菜降血压肽过程中的应用

为从紫菜蛋白中制备降血压肽,采用8种商业蛋白酶进行筛选,确定了以中性蛋白酶作为水解用酶和相应的酶解条件．研究了超声波处理紫菜蛋白后对酶解过程的影响,超声波处理后紫菜蛋白水溶液中游离氨基酸、酸溶性肽的质量浓度未变化,紫菜蛋白的紫外可见差示光谱也未出现变化;超声波处理的紫菜蛋白经酶解的产物,与未超声处理紫菜蛋白在同样条件酶解的产物相比,水解度增加1．1倍,IC50值降低29．5％．结果表明,超声处理没有破坏紫菜蛋白的分子结构,可能使它的分子空间构象发生变化,更加容易与酶分子结合,促进了酶解的进程．     

Aβ16—22聚集及海藻糖抑制其聚集过程的毛细管电泳检测

淀粉质β（amyloidp,Aβ）多肽是阿尔兹海默症（Alzheimer’Sdisease,AD）的致病蛋白,其疏水核心Aβ16—22具有重要的研究价值．以Aβ16—22为模型多肽,利用毛细管电泳检测方法,分析了Aβ16—22在水溶液和海藻糖溶液中的聚集过程．结果表明,毛细管电泳法不仅能够很好地分离Aβ16-22聚集过程中单体和部分聚体,实现对多肽聚集过程的快速鉴定和分析,同时也可检测海藻糖对Aβ16—22聚集的抑制作用．研究结果可为Aβ聚集抑制剂的高通量筛选和AD治疗中的快速诊断提供实验基础．     

文蛤蛋白水解制备抗氧化活性肽的研究

以文蛤蛋白为原料,通过蛋白酶水解制备抗氧化活性肽.利用体外清除DPPH活性评价酶解产物的抗氧化活性,采用超滤、葡聚糖凝胶柱层析和HPLC对相应的活性肽进行分离纯化,通过HPLC-MS进行结构鉴定.结果表明:木瓜蛋白酶水解产物的DPPH清除活性明显高于碱性蛋白酶、风味蛋白酶、中性蛋白酶和复合蛋白酶,产物质量浓度为1 mg·mL-1时,DPPH清除率为55.74%;以DPPH清除率为筛选指标,对木瓜蛋白酶酶解产物进行逐级分离,得到4个具有抗氧化活性的目的肽段,氨基酸序列分别为LNFNLEKSR(1120.3 u)、SWLRPR(814.4 u)、RIGNIISQY(1063.3 u)和LNFNLEK(877.2 u).     

4种促性腺激素释放激素抗体的制备、鉴定和纯化

用3-马来酰亚胺基苯甲酸琥珀酰亚胺酯(MBS)法将人工合成的4种不同来源的促性腺激素释放激(GnRH)(即章鱼GnRH、海鞘GnRH-Ⅰ、七鳃鳗GnRH-Ⅰ和七鳃鳗GnRH-Ⅲ)分别与匙孔戚血蓝蛋白(KLH)偶联后作为抗原,免疫新西兰大白兔获得GnRH抗体.用间接ELISA方法检测抗血清效价,并用Western blot鉴定其特异性.制备抗原肽亲和纯化柱,纯化多克隆抗体.结果显示,4种多克隆抗体效价高达1∶500 000(体积比),并能和相应的GnRH多肽特异性结合,纯化后的抗体在SDS-PAGE显示为单一条带.通过上述方法,获得4种高效价、高纯度的GnRH抗体,为低等动物GnRH的研究提供了理论依据.