脉冲响应技术在离子交换色谱中的应用

离子交换色谱法或脉冲响应技术提供了一个快速且经济的取代直接实验(TheConventional batch experiment)的方法,以建立平衡等温线,进而确定平衡常数.在低浓度溶液中,用脉冲响应技术估算的平衡常数 K 与直接实验法确定的这些参数有很好的一致性.     

用^211At和^131I标记蛋白质的研究

报道了砹—211和碘—131标记蛋白质的方法.通过过氧化氢或氯胺T直接氧化制备,使用凝胶色谱从反应产物中分离标记蛋白质,相对法比较放射性计数测定标记产率,过氧化氢适用于~(211)At标记蛋白质,氯胺T有利于~(131)I标记蛋白质.8次实验的结果表明,用过氧化氢氧化标记的~(211)At牛血清白蛋白产率96.4％,氯胺T氧化标记的~(?)I牛血清白蛋白产率66.1％.间接法标记蛋白质,放射性核素~(211)At同对氨基苯甲酸反应获得对—~(211)At—苯甲酸,经乙醚萃取和高效液体色谱分离,然后再偶联到免疫球蛋白或牛血清白蛋白上。动物实验结果表明,此种结合的标记蛋白质在体内稳定,标记率不低于初始~(211)At放射性活度的40%.     

裂褶菌的生物有效成分研究

本文对裂褶茵南大853菌株的有效成分进行了分析。在黄豆粉葡萄糖液体培养基中以30％的的葡萄搪浓度最有利于多糖得率的提高。在茵丝体和发酵液中均含丰富蛋白质和17种氨基酸,并富含8种必需氨基酸、茵丝体残渣和多糖中含相当量的蛋白质。茵丝体经等离子光谱法分析,含25种微量量元素,其中含有8种必需微量元素。     

The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

非直接荧光免疫法鉴定HMG＿（14）非组蛋白质在果蝇唾腺染色体上的原位分布

以获得的抗ＨＭＧ＿（１４）ＭｃＡｂ为探针,运用间接荧光免疫技术,对果蝇（Ｄ,Ｖｉｖｉｌｉｓ南京）唾腺染色体上ＨＭＧ＿（１４）的分布及其ＨＭＧ＿（１４）与活性区“ｐｕｆｆ”的关系进行了初步观察。发现;（１）ＨＭＣ＿（１４）分布在唾腺染色体所有的横纹带上;（２）在“ｐｕｆｆ”区,ＨＭＧ＿（１４）有增加的趋势;（３）ＤＮａｓｅＩ处理可以使染色体上的ＨＭＧ＿（１４）丢失。实验结果进一步验证了ＨＭＧ＿（１４）是染色体上的一种结构性蛋白。     

人表皮生长因子融合蛋白在大肠杆菌表达的研究

在大肠杆菌中高效表达人表皮生长因子(hEGF)。根据大肠杆菌对密码的偏爱性,构建高效表达EGF融合蛋白的菌株,经离子交换层析和凝胶过滤层析两个步骤使产物得到纯化。融合蛋白表达产量为27%,EGF融合蛋白纯化产物纯度为95%以上,促3T3细胞增殖的活性测定为ED50为4.2ng/mL。在pET 3c:BL21(DE3)大肠杆菌表达系统中,以融合蛋白方式表达人表皮生长因子,表达效率高,纯化路线简单,产量较高,适合产业化生产。     

百合属几种植物亲缘关系的可溶性蛋白质和过氧化物酶分析

采用聚丙烯酰氨凝胶电泳法，分析了百合属8种植物的可溶性蛋白质和过氧化物酶．结果显示：可溶性蛋白质谱共有48条带，其中26条带为多态性带；过氧化物酶谱共有20条带，19条带具有多态性．根据可溶性蛋白质和过氧化物酶的谱带进行了聚类分析，结果表明，生化水平与传统的形态学分类结果基本一致．     

水稻BpHi008A基因对褐飞虱取食应答及其编码产物在大肠杆菌中的高效表达

对抗虫相关蛋白BpH i008A进行了生物信息学分析,发现BpH i008A含有一个PKC磷酸化位点、一个CK2磷酸化位点、三个N-myristoylation site位点;另外,BpH i008A蛋白含有一跨膜螺旋,和位于细胞内的N-末端及伸向细胞外的C-末端.Northern分析证明褐飞虱的取食是维持BpHi008A基因的表达的必要条件.以pET28 a为载体构建了非融合表达的重组质粒,并且实现了BpHi008A基因在大肠杆菌菌株BL21(DE3)中的高效表达.     

红色荧光蛋白在毕赤酵母中的高效表达

报道了红色荧光蛋白(DsRFP)在毕赤酵母中的高水平表达．利用PCR技术从YEpFLAG-1-DsRFP扩增出DsRFP编码序列．克隆到毕赤酵母胞内表达载体pPIC3．5K构建重组表达载体pPIC3．5K-DsRFP．电击转化进毕赤酵母．G418-RDB平板双重筛选后获得重组转化子．经MM／MD平板培养与PCR鉴定．重组子全部为HIS^ -MUT^ 表型．重组菌株诱导表达48h后获得了高效表达．摇瓶中表达水平达到细胞内总蛋白的12％．表达量达到1．8g／L．经硫酸铵分级沉淀，DE-AE-5PW阴离子交换柱层析与S-HyperD阳离子交换柱层析后获得电泳纯蛋白．说明我们建立的毕赤酵母表达应用平台具有高效表达外源蛋白的能力。