类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

连续性血液净化在肾衰危重病人抢救中的应用及效果评价

目的:探讨连续性血液净化在危重病人抢救中的应用及效果评价.方法:选取2010年1月～2012年11月我院收治的危重病人共26例,均采用连续性静脉-静脉血液滤过方法治疗,比较治疗前后的心脏血流动力学参数、肾功能与电解质的变化,观察全部患者治疗前后的临床症状改善情况及临床转归.结果:治疗前后的心率、中心静脉压、平均动脉压、心指数及心排出量差异均存在统计学意义(P＜0.05);治疗后患者的血尿素氮、肌酐及钾水平均明显低于治疗前(P＜0.05);治疗后患者的低血压、低血氧、昏迷改善率分别为66.67％、71.43％及62.50％;26例患者中死亡14例(53.85％),存活12例(46.15％).结论:连续性血液净化可以积极有效地治疗危重病患者,选择治疗的时候需要针对患者的情况进行综合判断和全面评估.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

快速纯化重组腺联病毒的受体结合捕捉方法

【目的】建立用于重组腺联病毒(AAV)纯化的受体结合捕捉方法。【方法】将AAV受体的多囊肾病(PKD)结构域1和2与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,利用相变循环(ITC)纯化ELP-PKD融合蛋白;分别用昆虫和AAV-293细胞制备rAAV-GFP,与ELP-PKD融合蛋白共孵育后进行ITC,从沉淀复合物提取病毒DNA进行PCR检测;在优化条件下利用ELP-PKD蛋白结合捕捉rAAV-GFP,利用电子显微镜观察、免疫转印和细胞感染试验进行rAAV鉴定。【结果】ELP-PKD融合蛋白获得正确、可溶性表达,ITC纯化的蛋白纯度大于90%;ELP-PKD蛋白能特异结合rAAV-GFP,结合具有p H、温度和时间依赖性,受体结合捕捉方法可在1h内完成,从两种细胞纯化rAAV-GFP的回收率分别为58%和56%;rAAV-GFP洗脱具有p H和温度依赖性,洗脱rAAV-GFP的回收率分别为46%和44%;纯化rAAV-GFP具有AAV的典型形态和结构蛋白。【结论】建立的ELP-PKD结合捕捉法可用于不同细胞源rAAV的快速纯化。     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.