蝮蛇毒纤溶酶的分离纯化及性质研究

目的 :寻找一种分离纯化蝮蛇毒纤溶酶的工艺并研究其理化性质。方法 :采用DEAE SepharoseCL 6B和HeparinCL 6B层析方法 ,从蝮蛇毒中分离纯化纤溶酶。结果 :蝮蛇毒纤溶酶经HPLC为单一峰 ,等电聚焦电泳为一条带 ,其等电点为 4.5 5 ,经SDS 聚丙烯酰胺凝胶电泳测得分子量为 2 9.4kD。该酶对热不稳定 ,在 pH6～ 9时稳定 ,氨基酸组成分析表明含酸性氨基酸较多。结论 :用此工艺可制得高纯度的蝮蛇毒纤溶酶。     

山羊脾脏多肽的HPCE定性分析

目的：应用HPCE对由山羊脾脏中获得的多肽进行分析。方法：由经前期诱导后山羊脾脏中提取多肽；经毛细管等电聚焦电泳（CIEF）的方法测定其组成及各组分的等电点。结果：毛细管等电聚焦电泳测得山羊脾脏多肽各组分等电点单一，约为pI=4．46。结论：由山羊脾脏中获得的多肽是由等电点单一的成分组成。     

合成胸腺体液因子(THF-γ2)的纯度及结构鉴定

目的:对合成胸腺体液因子(thymus humoral factor,简称THF-γ2)进行纯度及结构鉴定。方法:用HPLC、毛细管区带电泳、质谱、氨基酸组成分析、序列测定和紫外扫描的方法鉴定THF-γ2的纯度和结构。结果:HPLC分析表明其纯度达到95.9%;毛细管区带电泳鉴定纯度为95.1%;质谱测定产物相对分子质量为917.7,与理论值918相符;氨基酸组成分析表明其氨基酸摩尔比与理论值一致;氨基酸序列测定结果为NH_2/Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu,与设计序列相同;紫外扫描除在252nm有Phe的微弱吸收,整体为一光滑曲线。结论:经检测合成THF-γ2纯度高于95%,鉴定了其结构与理论的一致性。     

治疗性单克隆抗体类药物分析技术及其应用

治疗性单克隆抗体(单抗)类药物是高度均一的生物大分子,抗原表位、理化性质和生物活性等方面的均一性程度高,能够通过特异性地与靶标分子结合发挥治疗作用。单抗类药物治疗过程中不良反应发生率低、患者耐受性高。单抗类药物是生物药物领域的热点和焦点,已成为现代生物制药行业中占比最大、增长最快的细分领域。根据生产技术和工艺的不同,单抗类药物发展已经经历了4代。我国最近批准上市的信迪利单抗注射液、特瑞普利单抗注射液、注射用卡瑞利珠单抗和替雷利珠单抗注射液等均属于第3代人源化单克隆抗体药物。单抗类药物分子结构复杂,容易发生二聚体、多聚体、末端氨基酸突变等不均一性变化,严重影响临床用药的安全性和有效性。对单抗类药物的原液和成品进行有效的质量控制是确保该类产品安全性和有效性的重要措施。目前,单抗类产品质量控制与分析的主要方法包括高效液相色谱(HPLC)法、酶联免疫吸附测定(ELISA)法、毛细管区带电泳(CZE)法、毛细管等电聚焦电泳(cIEF)法、成像毛细管等电聚焦电泳(iCIEF)法和十二烷基硫酸钠-毛细管电泳(CE-SDS)法等。本文就单抗类药物质量控制分析方法及应用进行综述,为相关研究及产品质量控制提供参考。     

蛇毒神经生长因子的进一步纯化与鉴定

对分离的蛇毒神经生长因子进一步纯化,并签定了其部分性质.结果纯化的蛇毒神经生长因子在浓度为5 ng/ml时仍有良好的生物活性。经SDS-PAGE分析,呈电泳一条带,其分子量约为14kD.等电聚焦电泳分析,等电点为6.80.紫外光谱分析,在220.6nm处有一个特异吸收峰,氨基酸组分分析表明由231个18种氨基酸组成。     

江浙蝮蛇毒镇痛组分的分离纯化及其理化性质

目的　分离筛选江浙蝮蛇毒中主要镇痛组分 ,研究其分子量、等电点、纯度、氨基酸序列、稳定性等理化性质 ,以及镇痛作用、机体耐受性和依赖性等药理学性质。方法　采用离子交换和分子筛 ,以柱层析法分离蛇毒组分。用生化测定方法 ,测定其理化性质。用热板法和醋酸扭体法等确定最佳镇痛组分 ,并用纳洛酮催促和自然戒断实验、耐受性实验考察其依赖性和耐受性。结果　分离获得 14个蛋白组分 ,经ED50 /LD50 筛选出最佳镇痛组分C4 ,鉴定结果表明纯度为电泳纯 ,HPLC相对纯度为 92 .16 % ,分子量 16 .6ku ,等电点 8.8,氨基酸序列与磷脂酶A2 同源 ,为一种新的镇痛蛋白 ,并获得其紫外吸收特征峰、镇痛耐受和依赖性等性质。结论　C4组分具有显著的镇痛效果 ,未发现耐受性和依赖性 ,值得进一步研究开发。     

大鲵皮肤分泌液中抗菌肽的鉴定及生物活性研究

目的鉴定大鲵皮肤分泌液中抗菌肽,研究其部分生物活性。方法 5%醋酸浸提和Sephadex G-50、G-25凝胶过滤色谱等方法分离纯化抗菌肽;采用抑菌圈法检测抗菌活性,Tricine-SDS-PAGE电泳和等电聚焦电泳鉴定其抗菌活性成份。结果大鲵皮肤分泌液中含有抗菌活性物质。对革兰阴性菌、革兰阳性菌和真菌均具有较强的抗菌活性;电泳检测显示该小分子多肽相对分子质量约为4 300,具有较强的碱性。结论首次从大鲵皮肤分泌液中分离纯化到一种抗菌肽,此抗菌肽可能是一个具有较强阳离子特征的碱性肽。     

合成胸腺体液因子（TIHF-γ2）的纯度及结构鉴定

目的：对合成胸腺体液因子（thymus humoral factor，简称THF-γ2）进行纯度及结构鉴定。方法：用HPLC、毛细管区带电泳、质谱、氨基酸组成分析、序列测定和紫外扫描的方法鉴定THF-γ2的纯度和结构。结果：HPLC分析表明其纯度达到95．9％；毛细管区带电泳鉴定纯度为95．1％；质谱测定产物相对分子质量为917．7，与理论值918相符；氨基酸组成分析表明其氨基酸摩尔比与理论值一致；氨基酸序列测定结果为NH2／Leu—Glu—Asp-Gly—Pro—Lys-Phe—Leu，与设计序列相同；紫外扫描除在252nm有Phe的微弱吸收，整体为一光滑曲线。结论：经检测合成THF-γ2纯度高于95％，鉴定了其结构与理论的一致性。     

辽宁产东亚钳蝎毒中哺乳动物毒素Ⅲ的分离纯化及部分性质的研究

采用 CM—Sephadex C—50,SP—Sephadex C—25离子交换柱层析和 SephadexG—50凝胶过滤三步分离程序,从东亚钳蝎毒中得到一种新的哺乳动物毒素。经低 pH 系统不连续聚丙烯酰胺凝胶园盘电泳,SDS—不连续聚丙烯酰胺凝胶板电泳及等电聚焦聚丙烯酰胺凝胶园盘电泳鉴定说明该毒素为电泳纯的蛋白质。用 SDS 电泳法测得其分子量为8,750道尔顿,等电聚焦电泳法测定 pH 为8.2。纯化成份的产率为2.5%。     

螺旋藻酸性多糖的电泳方法研究

利用琼脂糖凝胶电泳、聚丙烯酰胺梯度胶电泳及等电聚焦电泳对从螺旋藻中分离出的酸性多糖进行了研究。结果表明，螺旋藻酸性多糖的琼脂糖凝胶电泳呈单一斑点，与其他几种硫酸化多糖相比，迁移率有较大差异；利用聚丙烯酰胺梯度胶电泳可按分子质量大小将其分成3个组分，该结果与高效液相检测结果比较，两者基本吻合；螺旋藻酸性多糖的等电聚焦电泳呈单一条带，分布范围在pH值6．67～7．72。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.