单链抗体展示系统研究进展

单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。     

姜黄素对高氧致新生鼠支气管肺发育不良的保护作用

目的 研究姜黄素对高氧暴露致新生鼠支气管肺发育不良的影响,探讨其作用机制.方法 给予出生6 h内的SD大鼠持续60%氧暴露14d建立肺损伤模型.通氧的同时予姜黄索100 ms/(kg·d)灌胃.观察肺组织病理学改变,进行辐射状肺泡计数(RAC),末端脱氧核苷酸转移酶介导的dUTP缺口标记技术(TUNEL)检测肺组织细胞凋亡.免疫组化和Western blot法检测肺组织活化半胱氨酸蛋白酶-3(Caspase-3)的表达.结果 与空气对照组相比,随着氧暴露时间的延长,高氧组的大鼠出现肺发育停滞的典型病理表现:肺泡增大、结构简化,肺泡隔增厚.RAC明显减少,肺组织细胞凋亡明显增加,免疫组化和Western blot法均显示肺组织活化Caspase-3表达明显升高.姜黄素能改善损伤的肺病理结构,并在干预14d后使RAC显著增多,肺组织凋亡细胞显著减少,干预4d后肺组织活化Caspase-3显著降低.结论 姜黄素可减轻高氧暴露所致的BPD,可能是通过抗凋亡机制实现其保护作用.     

玉米液泡膜焦磷酸酶基因ZmVPP1的克隆及逆境下的表达分析

采用同源克隆的方法在玉米中得到一个与拟南芥耐盐基因AVP1类似的基因.BLAST分析表明,该基因编码蛋白属于质子泵焦磷酸酶家族(H_PPase superfamily)中的Ⅰ型VPP,将其命名为ZmVPP1.ZmVPP1包舍一个2301bp的开放阅读框,编码766个氨基酸残基.蛋白比对结果表明,该蛋白在不同植物中相当保守.实时荧光定量PCR检测发现,ZmVPP1基因在成熟叶片中高丰度表达,在生殖器官中表达量较少.脱水、高盐、低温等逆境胁迫条件和ABA处理下的表达分析表明,ZmVPP1基因受逆境的诱导表达,属于不依赖于ABA的途径.由此推断,ZmVPP1可能参与玉米对Na+的隔离,从而起到耐盐的作用.     

一株转化琼胶为新琼寡糖菌株的筛选及鉴定

摘要:【目的】筛选一株可以将琼胶转化为新琼寡糖的菌株,并对该菌株进行鉴定。【方法】从紫菜生长区域采集紫菜和该区域海水,用含1‰琼胶的培养基富集培养,逐级稀释涂布、平板划线进行初筛,液体培养进行复筛,DNS法测定琼胶降解产物中还原糖的含量。通过16S rDNA序列分析,结合菌体形态、菌落特征及生理生化特性,确立该菌的系统发育学地位。【结果】从紫菜振荡液中筛选出一株可以产琼胶酶的菌株HJPHYXJ-1,该菌属于革兰氏阴性菌,16S rDNA序列同源性与需钠弧菌(Vibrio natriegens)的达到了99%,结合形态特征和生理生化实验结果鉴定该菌为需钠弧菌。HPLC法测定酶解产物为新琼寡糖。【结论】HJPHYXJ-1被筛选用于转化琼胶,酶解产物的聚合度在2－12 之间,是以二糖为单位的新琼寡糖,该菌产生的酶为β-琼胶酶。     

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores'' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.     

黑龙江省黄鼬冬季毛被分层结构及保温功能

为了解黄鼬毛被的保温机制,选取黑龙江省通河林区黄鼬东北亚种(Mustela sibirica manchurica)冬季皮张,通过观测雄性5个部位和雌性4个部位毛被的分层结构,并结合热物性测试比对,发现了毛被的各层结构具有不同的保温隔热功能且存在部位差异。结果表明:1)毛被由外及里表现出4层结构,各层毛被厚度分别为:(12.7±3.0)mm、(6.0±1.8)mm、(5.5±2.2)mm和(1.4±0.5)mm。2)4层毛被的颜色、结构及保温机制依次为:最外层毛被棕黄色或金黄色,仅由2种类型被毛构成,耐磨损能力较强,保护下层毛被,阻挡冷空气侵入并降低毛被的热量散失;第二层毛被淡黄色,此层开始均由4种类型被毛构成,毛干细度较小,被毛间形成细小空隙,滞留大量静止空气,增强保温功能;第三层毛被灰色或灰白色,4种类型被毛的毛干更细,绒针毛和绒毛弯曲程度更大,使被毛间滞留静止空气的能力更强、更稳定,保温能力更强;最内层毛被为白色,为近毛根处,4种类型被毛均较直,便于热量传递。3)4层毛被的对整体的贡献率分别为:16.11%、27.40%、44.40%和12.09%。4)背面毛被较腹面的颜色深,厚度大,保温能力强;沿吻端至尾基的体轴方向毛被厚度增加,保温性增强。5)雄性毛被较雌性的厚度大,保温能力强。以上,反映了黄鼬冬季多部位毛被由表及里不同的空间布局及各层毛呈现的不同的形态结构,从整体上兼顾保护、保温、散热等多种功能,以适应当地的寒冷环境。     

X射线辐射对仔鼠消化酶活性及胃中Bax和Ghrelin表达的影响

为了探讨X射线辐射对仔鼠胃蛋白酶活性、十二指肠脂肪酶活性及胃中Bax蛋白和Ghrelin表达的影响,对170只仔鼠用不同辐射剂量（0,4,12,20,28 Gy）X射线进行全身辐射,分别在辐射后1,5,10,20 d用比色法检测仔鼠胃蛋白酶活性和十二指肠中脂肪酶活性的变化,用免疫组织化学方法检测胃中Bax蛋白和Ghrelin的表达和分布,并用Image-proplus 5.0专业图像分析软件检测Bax蛋白和Ghrelin在胃中的表达强度。结果表明,X射线辐射影响发育期仔鼠胃蛋白酶和十二指肠脂肪酶的活性以及胃中Bax蛋白和Ghrelin的表达。仔鼠胃蛋白酶活性除在辐射后1 d时高于对照组外,其它辐射后各期均低于对照组,仔鼠十二指肠中脂肪酶活性在辐射后均低于对照组;Bax蛋白主要在仔鼠胃黏膜上皮细胞中表达,其表达水平随辐射剂量的增大而增强;Ghrelin主要在胃内分泌细胞中表达,辐射后其表达水平降低。X射线辐射影响仔鼠消化酶活性,这可能与胃中Bax蛋白和Ghrelin的表达变化有关。     

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by ∼30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.The liver is the primary organ for carbohydrate and lipid metabolism, including gluconeogenesis, glycogenesis, cholesterol biosynthesis, and lipogenesis (1, 2). These metabolic events in the liver are tightly controlled by several pancreatic hormones including insulin and glucagon. In addition, the liver itself is one of the largest endocrine organs in the body, secreting numerous humoral factors involved in the regulation of systemic glucose and lipid homeostasis. The importance of the liver-derived humoral factors in maintaining glucose metabolism is highlighted by the observation that glucose uptake by skeletal muscle is severely impaired by surgical or pharmacological blockade of hepatic parasympathetic nerves (3). In the past several years, a number of liver-derived humoral metabolic factors, including bone morphogenetic protein-9 (BMP-9) (4), fibroblast growth factor 21 (FGF21) (5–7), retinol-binding protein 4 (RBP4) (8, 9), adropin (10), and angiopoietin-like proteins (Angptl) 3, 4, and 6 (11–13), have been identified, and their roles in glucose and lipid metabolism have been characterized in great detail. Noticeably, BMP-9, FGF21, and Angptl6 exhibit potent insulin-sensitizing and glucose-lowering effects in animal models, and they have been proposed as potential candidates for the treatment of insulin resistance and type II diabetes (4, 6, 7, 13).To search for novel liver-derived secretory factors involved in the regulation of glucose homeostasis, we used microarray analysis as a global screening for systematic identification of genes differentially expressed in the liver of C57BLKS db/db mice (a genetically inherited diabetic mouse model that is characterized by severe insulin resistance and hyperglycemia) and their lean littermates. We found that the mRNA level of mouse major urinary protein-1 (MUP-1)² was markedly down-regulated in db/db mice, and the change was largely normalized upon treatment with the PPARγ agonist rosiglitazone. MUP-1 is a small molecular weight secreted protein abundantly expressed in the liver (14). Its expression in the liver is enhanced by administration of the hepatotoxic agent dimethylnitrosamine (15) but is reduced by interleukin 6-induced acute phase response in mice (16). Like other members of the MUP family, MUP-1 has been proposed to act as a pheromone-binding protein in urine (17), thereby accelerating puberty and promoting aggressive behavior in male mice. However, the precise functions of MUPs have yet to be determined.MUP-1 belongs to the lipocalin superfamily, the members of which share a common tertiary structure with a cup-shaped hydrophobic ligand binding pocket surrounded by an eight-stranded β-barrel (18, 19). This structure confers upon lipocalins the ability to bind and transport a wide variety of small lipophilic substances, including fatty acids, cholesterols, prostaglandins, and pheromones. Noticeably, several members of the lipocalin family, including RBP4, lipocalin-2, and adipocyte fatty acid-binding protein (A-FABP), have recently been shown to be important mediators of obesity-related insulin resistance and glucose intolerance (8, 20–22). Unlike MUP-1, the expression of RBP4, lipocalin-2, and A-FABP are elevated in obesity and diabetes (9, 20, 23).In this study, we investigated the metabolic role of MUP-1 in mice. Our results demonstrated that MUP-1 was abundantly present in the circulation. In genetic and dietary obese mouse models, the serum and urine concentrations of MUP-1 were remarkably decreased. Replenishment of recombinant MUP-1 led to improved glucose tolerance and insulin sensitivity, as well as increased energy expenditure and locomotor activity in db/db diabetic mice. Our data suggest that MUP-1 not only serves as a circulating biomarker, negatively correlated with obesity-related metabolic disorders, but also plays an active role in regulating energy homeostasis and insulin sensitivity in mice.     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

Interaction with Tap42 is required for the essential function of Sit4 and type 2A phosphatases

In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase. The function of these phosphatases requires their association with different regulatory subunits. In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42. In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases. We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex. Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac. In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.