离子交换色谱流动相组成对溶菌酶复性的影响

目的 研究了复性缓冲液组成对还原变性溶茵酶复性的影响。方法用离子交换色谱法对还原变性溶菌酶进行复性。结果低离子强度的Tris缓冲液比高离子强度的PBS缓冲液能明显提高复性收率；当复性缓冲液中不合其他种类的盐时，脲浓度为2．0mol／L时复性产率最高；当脲浓度低时，复性缓冲液中含有氯化钠和硫酸铵的复性效果均不如不合这些盐的复性效果；当脲浓度高时，硫酸铵能很好地提高溶菌酶的复性回收率。结论认为Hofmeister效应是造成这些现象的主要原因。     

离子交换色谱流动相组成对核糖核酸酶的折叠影响

用离子交换色谱法（IEC）研究了还原变性核糖核酸酶（RNase A）折叠过程中，复性缓冲液种类及pH值、流动相中脲浓度及盐种类对还原变性核糖核酸酶复性的影响。发现pH为弱碱性，用PBS作为缓冲液能提高复性效率；当流动相中含有2．0mol／L脲时复性产率较高；洗脱剂用NaCl时活性回收率较高。     

添加剂促进变性还原溶菌酶复性的作用

研究了多种添加剂促进变性还原溶菌酶复性的作用，考察了添加剂浓度及变性剂盐酸胍浓度对复性收率的影响．结果表明，精氨酸、乙酰胺、丙酮、硫脲及甘油均能有效促进变性溶菌酶复性，并且存在最佳的添加剂浓度使变性溶菌酶的复性收率最大．在促进复性中，乙酰胺等结构类似物与盐酸胍具有相同作用，因此，在降低复性液中盐酸胍浓度的同时，适当提高乙酰胺浓度即可获得较高的复性收率．当盐酸胍浓度为0．2mol／L时，复性收率达到90％时的乙酰胺浓度为2mol／L，但降低盐酸胍浓度至0．06mol／L时，达到相同变性收率的乙酰胺浓度需要4mol／L．甘油与盐酸胍存在着协同作用，在一定浓度的盐酸胍存在下，添加适量的甘油能获得较高的复性收率．     

基因工程血管抑素3A蛋白的分离纯化

在8mol／mL尿素溶液中,以二硫苏糖醇(DTT)为还原剂,对基因工程血管抑素3A包涵体进行溶解实验。结果发现：1．5h后溶解的上清液蛋白浓度达26．2mg／mL。SDS-PAGE电泳扫描结果显示,3A蛋白经过Sephadex G75分离后PAGE电泳纯达到100％,该步收率达85．82％,相对分子质量为10ku。采用分步稀释法对其进行复性,所获复性3A蛋白经MTT法检测表明：3A蛋白浓度为0．1μg／mL时,对内皮细胞的生长抑制率为93．4％。     

Prokaryotic expression and characterization of a pea actin isoform （PEAcl） fused to GFP

Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells,it is difficult to purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions in 8 mol/L urea,renaturing by dialysis in a gradient of urea,and affinity binding to Ni-resin. The purified mono-meric PEAc1-GFP could efficiently bind on DNase I andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.     

Increase Renaturation Yield of Reteplase Using the Recombinant Human Protein Disulfide Isomerase

Reteplase, the recombinant type of novel tissue plasminogen activator (t-PA) variant, is a promising thrombolytics in clinics. Expressed in the form of an inclusion body, reteplase consists of about 40% of the total intracellular proteins of Escherichia coli. The recombinant human protein disulfide isomerase (rhPDI) is used to increase the chance for the correct matching of the 18 hydrosulfide groups of the reteplase molecule in the renaturation process and it increase is the reteplase renaturation yield from 1%-2% to 15% - 20% with a the purity about 99% and the specific activity of 5(105 IU/mg is reached.This novel method can reduce significantly the cost of production.     

3种杀虫剂对杨树羧酸酯酶的抑制效果

研究了酶浓度、底物浓度、反应体系pH、反应温度、反应时间5个因素对美洲黑杨羧酸酯酶(CarE)活性测定的影响,得出测定杨树CarE的最适反应条件为:酶稀释10倍,底物浓度为3×10-4mol/L,pH为7.0,温度30℃,时间15 min。利用试验得到的反应最适条件分析了氧化乐果、吡虫啉和氟虫腈3种内吸性药剂对杨树CarE活性的影响。结果表明:400、1 600、3 200和6 400mg/L氧化乐果能明显抑制杨树CarE活性,其中6 400 mg/L氧化乐果抑制作用最强。50、100、200、400和800 mg/L吡虫啉处理杨树48 h,对杨树CarE均没有显著影响;当处理96 h时,400和800 mg/L吡虫啉能显著抑制CarE活性。而400 mg/L氟虫腈处理杨树48 h,800 mg/L氟虫腈处理杨树96 h,都能显著抑制杨树CarE活性。     

耐盐性层析吸附剂的制备及性能

以再生纤维素钛白粉复合微球作层析基质,经环氧活化后实施两步功能基化反应,同时引入疏水基团和离子交换基团,制备了一种耐盐性层析吸附剂。其蛋白质吸附性能研究表明:吸附剂的吸附能力不依赖于盐浓度,对含有0.25 m o l/L N aC l的蛋白质溶液的平衡吸附容量分别为:牛血清白蛋白27 m g/mL,溶菌酶40 m g/mL。     

Antibacterial Effects of Silver Loaded Hydroxyapatite

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人P53的原核表达及包涵体复性研究

通过检测血清中p53蛋白及其抗体变化诊断肿瘤的ELISA试剂盒的研发需要大量制备p53蛋白.本研究通过构建人野生型p53基因的原核表达质粒pET-32a-p53,在大肠杆菌中诱导表达得到p53包涵体蛋白,经包涵体变性、亲和纯化、复性得到可溶p53蛋白.发现利用8M尿素对包涵体进行变性溶解,经镍柱亲和纯化得到纯度超过95%的变性蛋白.尝试透析复性、稀释复性和柱上复性3种方法分别对p53变性蛋白进行复性.结果表明透析复性的复性率最高,这为原核表达制备p53提供了一种参考.本研究为ELISA法检测血清中p53蛋白及其抗体诊断肿瘤相关试剂盒研发奠定了基础.