药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

重组干扰素-τ在大肠杆菌中的表达与纯化

目的:探讨干扰素(IFN)-τ在大肠杆菌中的表达及纯化。方法:含有IFN-τ基因的pBV220表达质粒转化大肠杆菌BL21,于42℃温控诱导重组菌表达IFN-τ。经过包涵体溶解、DEAE离子交换层析、硫酸铵沉淀及梯度透析,使重组IFN-τ获得纯化和复性。结果:诱导后的表达产物经SDS-PAGE分析,有相对分子质量约21000的条带。纯化复性后目的蛋白纯度可达90%。结论:工程菌可稳定高效地表达IFN-τ。硫酸铵沉淀结合阴离子交换是一种简便高效的纯化方法,可获得较高纯度的IFN-τ。     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

用超滤法从猪血中分离纯化猪血红蛋白

为了给人工红细胞的研究提供较高纯度和浓度的血红蛋白,采用超滤膜分离的方法,通过研究分析,在一定超滤条件,从新鲜猪血中分离纯化得到猪血红蛋白。并应用SDS-PAGE凝胶电泳、紫外-可见分光光度计对纯化后的血红蛋白进行了鉴定分析。经分析,优化的超滤条件为:操作压力0.05Mpa、料液温度12℃;在此条件下分离纯化的猪血红蛋白,纯度达到了99%以上,浓度较纯化前提高了近20倍。     

Beta proteobacterium sp.T1菌株产壳聚糖酶的分离纯化

目的:分离纯化Beta proteobacterium sp.T1菌株所产的壳聚糖酶.方法:采用(NH4)2SO4(20%～70%)分级盐析、阴离子交换树脂DEAE Cellulose 52柱层析、SephadexG-100柱层析技术进行分离纯化,采用SDS-PAGE鉴定酶的纯度、分子量.结果:经DEAE Cellulose 52柱层析,壳聚糖酶纯化了13.19倍;经Sephadex G-100柱层析,壳聚糖酶纯化了26.32倍.结论:纯化后的酶经SDS-PAGE鉴定已达到电泳纯,分子量29.5kDa.     

米曲霉F-81菌株产中性蛋白酶的分离纯化

对米曲霉菌种F-81所产中性蛋白酶进行分离纯化。经过硫酸铵分级沉淀,DEAE-Sepharose Fast Flow阴离子交换层析和Sephacryl-S200凝胶过滤层析后,得到一种电泳纯的中性蛋白酶,纯化倍数为26.3倍,活性回收率为6.7%。经SDS-PAGE电泳测定其相对分子质量约为73.4kD。     

辛酸沉淀结合阴离子交换层析纯化马IgG的研究

目的以辛酸沉淀结合离子交换层析纯化破伤风抗毒素马免疫血浆,获得高质量的马IgG,为抗毒素F(ab')2的制备奠定基础。方法通过对辛酸沉淀马血浆过程中的pH、辛酸浓度以及血浆稀释倍数的实验设计(DoE),研究不同条件对IgG纯度、效价、比活性、浊度以及过滤速度的影响,确定各工艺参数的可操作区间,并结合Capto DEAE阴离子交换层析以流穿模式进一步纯化IgG。结果马血浆经一步辛酸沉淀获得的IgG纯度(SDSPAGE)大于92%、分子排阻色谱(SEC)纯度大于95%,比活性较血浆提高2.14±0.29倍;辛酸沉淀后的IgG样品经阴离子交换层析,可有效去除聚合体以及小分子杂质,将纯度提高至95%(SDS-PAGE)和98%(SEC)。结论马血浆经辛酸沉淀和阴离子交换层析可获得高纯度、高比活的IgG。     

超滤过结合密度梯度离心纯化鲍曼不动杆菌噬菌体方法的建立

目的 探讨获得低浓度内毒素和高滴度鲍曼不动杆菌噬菌体的方法,为制备安全的噬菌体生物制剂提供参考.方法 用可截留100 kD以上分子量的超滤离心管浓缩噬菌体裂解液并滤出分子量约为10 kD的内毒素,然后用蔗糖密度梯度离心纯化噬菌体浓缩液;分别测定超滤前、超滤后和纯化后的噬菌体滴度,采用鲎试验测定超滤前后内毒素的浓度,通过SDS-PAGE分析超滤前后和纯化后噬菌体蛋白的纯度.结果 经超滤离心法噬菌体滴度从3.9×1010 PFU/mL提高至1.68×1012PFU/mL,并可去除99.2％的内毒素;超滤过结合密度梯度离心后的SDS-PAGE可清晰呈现7种蛋白,分子量为29～100 kD.结论 超滤过结合密度梯度离心是一种简便、快速浓缩和纯化噬菌体的方法,并可有效地去除裂解液中的内毒素.     

重组A型产气荚膜梭菌α毒素C端片段的分离纯化

押在构建出高表达、高活性的A型产气荚膜梭菌α毒素保护性抗原工程菌株pBV/cpa408的基础上,对表达产物进行了分离纯化研究。诱导表达菌液离心后,超声破碎沉淀细菌,上清用80%饱和硫酸铵沉淀,沉淀蛋白经透析,上凝胶过滤层析柱分离纯化,得到纯度达95%以上的表达目的蛋白,经SDS-PAGE测定,相对分子质量为15.5×103,序列与文献报道的相符。     

Verification of the final anion exchange chromatography in the r-hGH manufacturing process

In this study, final anion exchange chromatography in the recombinant human growth hormone (r-hGH) manufacturing process was validated using a validation protocol that was consistent with both policy and standard operation procedure (SOP). Two buffer solutions used in chromatography were first validated and were found to satisfy pre-established acceptance criteria as follows: pH: 8.2, endotoxin: < 0.6 EU/mL, bioburden test: negative. Final anion exchange chromatography was conducted using a DEAE Sepharose FF Resin and eluted with a linear gradient of 30 to 110 mM NaCl in 50 mM Tris-HCl buffer at a flow rate of 15 L/h. Three consecutive batches of hGH solutions were generated via anion exchange chromatography, which was performed within pre-established operating parameters determined through in-process control. When all three batches were assessed by the pre-established sampling plan and tested for quality control, this purification process was shown to satisfy pre-established acceptance criteria; endotoxin: ≤ 0.5 EU/mg, ECP: ≤ 1.4 ppm, IEF: same removal distance, hGH content by Native-PAGE: 100%, purity by HPLC: ≥ 99%, yield by UV scanning: 87 to 89%, hGH monomer protein content by HPLC: 99%. Therefore, the final anion chromatography process was successfully validated in this study, and this method consistently yielded hGH solutions that satisfied pre-established criteria for subsequent processing.     

Purification of 120-Kilodalton Phytochrome from Oryza Sativa L

The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiolated rice (Oryza sativa L. subsp, japonica var. nongken 58) seedlings. Approximately l kg of 6d old seedlings (the first 2 days at 33℃, the last 4 days at 27 ℃ in darkness) were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer, at final pH 8.45 (4 ℃). After polyethylenimine precipitation, phytochrome in extract was converted to Pfr by irradiation of the resulting supernatant for 10 min with red light. The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ratio of 10 ml per phytochrome unit. The pellet precipitated with ammonium sulfate at 42% saturation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8 ml instead of 0.65 ml per phytochrome unit. Then it was washed successively with 200 mmol/l and 100 mmol/1 phosphate buffer (0.85 ml per phytochrome unit). Native phytochrome (120 kDa) in 12% yield was dissolved in 2 mmol/l EHPES buffer (2.2 ml per phytochrome unit, pH 7.8, containing 5 mmol/l EDTA and 14 mmol/l 2-mercaptoethanol) was proved to be pure in SDS- polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome.     

白腐真菌漆酶的纯化及性质

液体发酵培养白腐真菌F9,粗酶液经盐析、透析浓缩、葡聚糖G-100柱层析、DEAE-纤维素离子交换层析四步分离纯化,得电泳纯漆酶。经SDS-PAGE法测定酶的相对分子质量约为6×104,酶活回收率达46.47%,纯度提高了18.86倍。F9漆酶最适反应温度为40℃,最适反应pH为4.8,在35℃以下、pH 4.8～5.4的范围内稳定性较强。其催化愈创木酚的Km为4.61 mmol/L,vm为6.27 mmol/(L.min)。K+对其有激活作用,而Fe2+、Fe3+对其有明显抑制作用。     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

离子交换层析结合反向色谱纯化聚二氨基丙酸

ε-聚赖氨酸生产菌株Streptomyces albulus PD-1可合成一种新型非蛋白质氨基酸均聚物聚二氨基丙酸,采用离子交换层析和反向色谱,对聚二氨基丙酸的分离纯化进行研究。离子交换层析柱选用DEAE-Sepharose Fast Flow填料,50 mmol/L磷酸盐缓冲液(p H 7.5)平衡上样,含0.5 mol/L Na Cl的磷酸盐缓冲液(p H 7.5)洗脱,收集洗脱液用分子筛Sephadex G-25除去磷酸盐缓冲液。然后用C18反相色谱进一步纯化,流动相为V(甲醇)/V(0.1%磷酸)=5/95。经过离子交换层析和反向色谱,纯化得到聚二氨基丙酸纯品,回收率为39.8%,样品纯度达98.4%,为后续的聚二氨基丙酸的深入研究奠定基础。     

猪圆环病毒2型病毒样颗粒的高效组装技术研究*

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。     

小鼠肠道菌群HPLC分析中色谱分离条件的优化

研究了在小鼠肠道菌群的高效离子交换色谱（HPLC）分析中,不同色谱分离条件对分离效果的影响,确立了最佳色谱条件：样品上样于Toyopearl TSKgel SuperQ-650c强阴离子交换树脂柱,以0.02mol/L哌嗪-HCl缓冲液（pH8.0）平衡,洗脱盐浓度为1.0mol/L NaCl,洗脱梯度为0.1-0.5mol/L NaCl线性梯度洗脱80min,再经0.5-0.75mol/L NaCl线性梯度洗脱25min,流速1mL/min,进样量为1mL。小鼠肠道菌群HPLC分析方法的建立,为深入研究小鼠肠道菌群的组成和动态变化奠定了基础。     

Purification and characterization of human hemoglobin: effect of the hemolysis conditions

Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 degrees C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.     

新的肝细胞生长因子HPPCn的表达、纯化及体外活性检测

目的：建立rhHPPCn在大肠杆菌中的表达体系,纯化表达产物并检测其体外生物活性。方法：以pUC18-HPPCn为模板,构建原核表达载体pET-24a(+)-HPPCn,转化到大肠杆菌BL21中并诱导表达目标蛋白,离子交换色谱法纯化目标蛋白,Western blot分析其免疫原性,MTT法和3H-TdR掺入法检测其体外活性。结果：成功构建出原核表达载体pET-24a(+)-HPPCn,并诱导使目标蛋白的表达量较高,表达产物中HPPCn 23％以可溶形式存在,两步离子交换后可得纯度为94.2％,Western blot鉴定为目的蛋白,MTT法和3H-TdR掺入法检测其具有明显的促肝癌细胞系增殖活性。结论：成功构建出原核表达载体并实现了目标蛋白高表达,两步离子交换法纯化出所需纯度的蛋白,并验证了其较强的体外促肝癌细胞系增殖活性。     

灰绿曲霉β-葡萄糖苷酶的分离及特性

目的：利用灰绿曲霉EU7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法：灰绿曲霉EU7-22发酵液离心后,上清液经硫酸铵沉淀、Phenyl 6 Fast Flow（highsub）疏水层析和Sephacryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果：纯酶的比活性为5.1 IU/mg,得率为13.89%。SDS-PAGE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kDa。在pH4.0~6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应pH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 h后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 h后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论：灰绿曲霉EU7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。