H5N1亚型禽流感病毒NS1基因截短体的克隆与原核表达

禽流感病毒H5N1 NS1蛋白是一种非结构蛋白,在病毒感染过程中发挥着重要的作用.构建基因截短的重组蛋白,可为进一步研究NS1不同结构域与宿主蛋白间的相互作用奠定基础.在成功克隆禽流感病毒H5N1全长NS1基因并测序的基础上,将部分截短基因序列克隆到表达栽体pET28a(+)上,构建基因截短的重组表达质粒pET28a-NS1-RBD和pET28a-NS1-ED,转化大肠埃希菌BL21(DE3),阳性重组质粒经IPTG诱导表达后进行SDS-PAGE检测,获得预期蛋白的表达,然后利用Ni-NTA树脂蛋白纯化系统对重组蛋白进行纯化,并通过Western Blotting进一步确认NS1及截短体蛋白的表达.结果表明,实验成功构建禽流感病毒H5N1亚型的NS1蛋白截短体,并在大肠埃希菌中高效表达,这为进一步研究NS1蛋白不同结构域与宿主蛋白的相互作用提供了实验材料,为深入研究NS1蛋白的生物学功能奠定了坚实基础.     

刺参“腐皮综合症”致病菌LNUB415的分离及防治的初步研究

从大连海域患“腐皮综合症”的养殖刺参肠道中分离纯化到1株优势细菌LNUB415,经人工回接感染试验确定其对刺参具有较强的致病性.经形态学、生理生化和16S rDNA序列分析,该菌被初步鉴定为蜡样芽胞杆菌(Bacillus cereus).从多种具有免疫增强作用的中草药中筛选到抑菌效果最好的黄连,可使刺参死亡率降低30％.     

Aβ在APP/PS1双转基因与APP/PSl/Tau三转基因阿尔茨海默病模型小鼠脑海马区分布的比较研究

目的:比较研究Aβ免疫阳性产物在老年APP/PSl双转基因(2xTg)与APP/PSl/Tau三转基因(3xTg)阿尔茨海默病模型小鼠海马的分布。方法:采用15月龄2xTg与同龄3xTg,分别进行Aβ单克隆抗体6E10免疫组化检测Aβ阳性神经元及斑块。结果:在海马区2xTg组Aβ沉积多发生于细胞外,形成大量Aβ阳性斑,而3xTg组则主要沉积于细胞内。结论:这种Aβ分布的差异可能与3xTg模型早期即有神经元丢失有关。     

Generation of anaphylatoxins by human beta-tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.     

玉米根系分布空间和空间密度分布的数学模型

应用重积分研究了土壤层中散根型和直根型玉米根系的分布空间,同时研究了玉米根系的空间密度分布．     

具有一般形式饱和接触率SEIS模型的周期解

利用重合度的延拓定理,导出了具有一般形式饱和接触率SEIS模型周期解的存在性准则.     

祁连山3种典型生态系统土壤微生物活性和生物量碳氮含量

 测定分析了祁连山高寒草甸、山地森林和干草原土壤中微生物活性、生物量碳氮含量。结果显示：就土壤微生物生物量碳含量,森林比干草原和高寒草甸中分别高60%和120%以上,干草原比高寒草甸中高40%以上(p<0.05)。就土壤微生物生物量氮含量,0～5 cm土层,森林比高寒草甸和干草原中分别高64%和111%以上,高寒草甸比干草原中高29%;5～15 cm土层,森林比干草原和高寒草甸中分别高7%和191%以上,干草原比高寒草甸中高171% 以上(p<0.05)。森林和干草原中土壤微生物生物量碳比例比高寒草甸中高32%以上,0～5和5～15 cm土层,森林和干草原中土壤微生物生物量氮比例比高寒草甸中高150%以上（p<0.05）。就土壤微生物活性,0～5和5～15 cm土层,森林和高寒草甸比干草原中高26%以上;15～35 cm土层,森林比干草原和高寒草甸中高28%以上 (p<0.05)。土壤微生物生物量碳氮含量与有机碳含量及微生物生物量氮含量和比例与微生物生物量碳含量和比例呈现正相关（r²>0.30,p<0.000 1）。土壤微生物生物量氮含量、微生物生物量碳氮含量比例、微生物活性与土壤pH值呈显著负相关,土壤微生物生物量碳氮含量及其比例、微生物活性与土壤湿度呈正相关。说明祁连山3种生态系统土壤中微生物生物量和活性受气候要素、植被、有机碳、pH值和湿度等因素 的共同影响。     

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.     

African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K (hnRNP-K) during infection

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as interacting cellular protein with the abundant immediate early protein p30 from African swine fever virus (ASFV) in a macrophage cDNA library screening. The interacting regions of hnRNP-K with p30 were established within residues 35-197, which represent KH1 and KH2 domains responsible for RNA binding. Colocalization of hnRNP-K and p30 was observed mainly in the nucleus, but not in the cytoplasm of infected cells and infection modified hnRNP-K subcellular distribution and decreased the incorporation of 5-fluorouridine into nascent RNA. Since similar effects were observed in cells transiently expressing p30, this interaction provides new insights into p30 function and could represent a possible additional mechanism by which ASFV downregulates host cell mRNA translation.     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.