一种高效、稳定的分泌型原核表达载体的构建及应用

以本室构建的原核表达载体pTO-T7为基础载体，PCR合成ompT引导序列，插入该载体多克隆位点上游，构建了分泌型原核表达载体pTO-OT．将2个外源基因克隆至pTO-OT，2个重组质粒在大肠杆菌中均得以高效表达．表达产量在25％～34％之间．Western blot分析证实了融合蛋白可被大肠杆菌信号肽酶有效地切割。并具有良好的免疫学活性．对重组表达菌株的连续传代实验证实了该表达载体具有良好的表达稳定性，显示了其在基因工程中的应用价值．     

U73122和三氟吡啦嗪对柴胡皂甙(I)促胰腺腺泡酶分泌的影响

应用碘淀粉比色法检测淀粉酶活性技术 ,以卡巴可 ( CCh)及八肽胆囊收缩素 ( CCk-8)为阳性对照药 ,研究了磷脂酶 C( PLC)抑制剂 U731 2 2和钙调素 ( Ca M)抑制剂三氟吡啦嗪 ( trifluoperazine)影响柴胡皂甙 ( I) [SA( I) ]刺激大鼠胰腺腺泡酶分泌的剂量依赖性和分泌动力学特征 .U731 2 2抑制 SA( I)和 CCh促酶分泌有相似的剂量依赖性 ,1 0 - 5mol·L- 1 U731 2 2分别抑制了相同浓度 SA( I)和 CCh作用的 5 7.4 %和 6 1 .5 % .U731 2 2抑制 SA( I)促酶分泌动力学较对 CCh的抑制缓慢且持续时间较长 .trifluoperazine抑制 SA( I)促酶分泌与对 CCk-8的抑制有相似的剂量依赖性 ,高浓度对 SA( I)作用的抑制更显著 .结果表明 ,SA( I)活化 PLC和 Ca M包括在其促酶分泌的细胞信号传导通路中     

葡萄糖供应速率对大肠杆菌表达及分泌人表皮生长因子的影响

采用一株大肠杆菌BL 21(DE 3)[pBLMVL 2EGF]生产人表皮生长因子(hEGF),其中hEGF表达由PL启动子控制并通过phoA信号肽分泌到胞外。实验结果表明:诱导阶段以恒定比供应速率流加葡萄糖时,流加速率对hEGF的表达和分泌有很大影响。当葡萄糖比供应速率为0.122g/(g.h)时,hEGF表达水平最低,分泌效率最差,只有37.5%;随着葡萄糖比供应速率的增加,胞外和胞内hEGF比生产速率明显增加,当葡萄糖比供应速率为0.196 g/(g.h)时,单位菌体产生的hEGF水平最高(121.6 m g/g),其中分泌至胞外的hEGF占42%。此外,诱导前补充有机氮源有利于提高诱导初始hEGF的生产速率。     

Rosiglitazone counteracts palmitate-induced β-cell dysfunction by suppression of MAP kinase, inducible nitric oxide synthase and caspase 3 activities

Chronic exposure of pancreatic islets to elevated levels of palmitate leads to beta-cell dysfunction. We examined possible involvement of mitogenactivated protein kinases (MAPKs) and caspase-3 in palmitate-induced beta-cell dysfunction and tested the influence of the anti-diabetic drug rosiglitazone (ROZ). Palmitate amplified glucose-stimulated augmentation of intracellular free calcium ([Ca2+]i) and insulin secretion in incubated islets. ROZ suppressed this amplification, whereas it modestly augmented glucose-induced increase in these events. ROZ suppressed short-term palmitate-induced phosphorylation of pro-apoptotic MAPKs, i.e., SAPK/JNK and p38. Long-term islet culturing with palmitate induced inducible nitric oxide synthase (iNOS) and activated SAPK/JNK-p38. ROZ counteracted these effects. Both palmitate and cytokines activated caspase-3 in MIN6c4-cells and isolated islets. ROZ suppressed palmitate- but not cytokine-induced caspase-3 activation. Finally, after palmitate culturing, ROZ reversed the inhibitory effect on glucose-stimulated insulin release. We suggest that ROZ counteracts palmitateinduced deleterious effects on beta-cell function via suppression of iNOS, pro-apoptotic MAPKs and caspase-3 activities, as evidenced by restoration of glucose-stimulated insulin release.     

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A

Catestatin (bCGA_344–364), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA_344–358) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA_344–358 in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA_344–358) (named cateslytin) as a novel component of innate immunity.Received 21 October 2004; received after revision 6 December 2004; accepted 7 December 2004     

一种基于粒度的规则挖掘方法

利用知识的粒度,提出了一种新的规则挖掘方法.首先利用粗糙集中判断属性重要性的尺度,获取一个属性,由此属性形成对域的粒度,并对粒度中的每一个粒进行同样操作,直至所有粒的决策属性值唯一为止.算法分析与实验对比说明,该方法是有效可靠的.     

物料颗粒度及平均灰度的特征提取

在物料的计算机自动识别过程中,颗粒度及平均灰度特征对物料的识别率非常重要 。本文通过对两者的研究,给出了二者的提取方法     

小儿康冲剂的药效学研究

为考察小儿康冲剂的健脾开胃、消食导滞、安神定惊功效 ,用健脾、消食、止痛、安神等方法对小儿康冲剂进行了药理研究 .结果表明 :小儿康冲剂可明显对抗大黄煎液和利血平引起的小鼠脾虚性体重下降和体温降低作用 ,并明显提高正常大鼠胃蛋白酶活力和促进小鼠小肠运动 ,减少小鼠自发活动和增加戊巴比妥钠诱导的小鼠入睡百分率 ,同时具有延长小鼠游泳时间和提高小鼠耐缺氧能力 .提示该药具有健脾、扶正、消食、安神和止痛之功效     

Ⅱ型糖尿病发病机制的研究进展

Ⅱ型糖尿病是由遗传和环境因素相互作用而引起的一组代谢异常综合征。其发病机制复杂,目前病因尚不明确,现能确定的是胰岛素抵抗和胰岛素分泌缺陷是Ⅱ型糖尿病发病的主要环节。     

正交试验法在颗粒材料堆自组织分析中的应用

颗粒材料堆具有非均质性、非连续性及随机性等内在的独特性质,从而给研究带来很大困难.颗粒材料堆在承载时,其力学性质与颗粒的大小、排布、堆积高度、表面的粗糙程度以及加载速度都有着密切关系.通过采用室内正交实验,利用自制的设备,研究了堆积高度、粒径和加载速度3个因素在不同水平下对颗粒材料堆自组织现象的影响.