人C1酯酶抑制剂初步纯化过程pH值和聚乙二醇含量的优化#br#

目的 探讨pH值、聚乙二醇（polyethylene glycol，PEG）含量和离心条件对PEG4000粉末用于去除人C1酯酶抑制剂（C1 esterase inhibitor，C1-INH）制备原料中的IgM效果的影响，并通过对羧甲基（carboxymethyl，CM）离子交换层析中洗脱盐离子浓度的筛选，分离活性与非活性C1-INH。方法 向不同pH值的C1-INH制备原料中加入不同质量分数的PEG4000，于不同条件下离心后，用特定蛋白检测仪对离心后上清液中IgM和C1-INH含量进行检测，确定PEG沉淀法纯化C1-INH的最佳条件。将离心后上清液调节pH后作为CM离子交换层析上样样品，使用不同盐离子浓度的洗脱液对活性C1-INH进行分离，确定最佳的盐离子浓度。结果 在弱酸性pH（6.8）下，当PEG4000质量分数为12%，离心条件15 000×g、25 ℃、20 min时，IgM去除率>99%，且C1-INH的回收率>80%；在盐离子浓度为200 mmol/L时，产物中C1-INH的比活性最大（4.43 IU/mg），且绝大多数杂质蛋白得以去除。结论  优化条件下PEG4000能有效去除C1-INH制备原料中的IgM，且能保持较高的C1-INH回收率；CM离子交换层析能对活性与非活性C1-INH进行有效分离，并去除大多数杂质蛋白。     

聚乙二醇化海葵神经毒素的修饰反应条件及药效研究

目的优化聚乙二醇(PEG)修饰海葵神经毒素rhk2a(rhk2a)的反应条件,并考察修饰后产物PEG化海葵神经毒素rhk2a(mPEG-rhk2a)的药效性质。方法本研究选用分子量为5 000的PEG丙醛(mPEG-ALD)对rhk2a的N-末端氨基进行修饰;通过单因素考察和正交试验筛选出最佳的修饰反应条件。同时,采用HPLC检测修饰后产物中mPEG-rhk2a的纯度,并通过建立急性心力衰竭豚鼠模型,考察mPEG-rhk2a对豚鼠的强心作用。结果优选出的mPEG-ALD对rhk2a的N-末端氨基的修饰反应条件是:当pH为5.0、rhk2a与mPEG反应摩尔比1∶20、还原剂氰基硼氢化钠为30μL、反应时间为12 h时,所得修饰产物mPEG-rhk2a的得率较高。采用SP-650S强阳离子交换层析法对修饰反应后产物进行分离,纯化后产物中mPEG-rhk2a占83.287%。对于心衰豚鼠,给mPEG-rhk2a溶液后,左心室收缩压、左心室压力最大下降速率明显增加,20 min后呈持续的平稳状态。其强心作用虽然不如rhk2a强,但波动起伏较小,而且强心效果明显高于乙酰毛花苷注射液。结论优选出mPEG修饰rhk2a反应的适宜条件,所得的mPEG-rhk2a强心作用较为理想。     

Box-Behnken设计-响应面法优选复方三七降脂滴丸成型工艺

摘要：目的:优选复方三七降脂滴丸成型工艺。方法:运用Box-Behnken中心组合设计试验,在单因素试验的基础上,以滴速、滴制温度、滴距为自变量,滴丸的溶散时限、重量差异、外观评分的总评归一化值（OD）为因变量,利用响应曲面法进行分析,选出最佳成型工艺条件并进行验证试验。结果:复方三七降脂滴丸最优成型工艺条件为：PEG4000与PEG6000配比为3∶1,药物与基质配比1∶4,滴速为30～40滴/min,滴制温度80℃,滴距4 cm。结论:本试验采用Box-Behnken设计-响应面法优选的复方三七降脂滴丸成型工艺方法稳定、可行。     

聚乙二醇修饰天花粉蛋白的初步研究

目的:研究聚乙二醇(PEG)修饰天花粉蛋白(TCS)的条件、纯化方法、生物活性及致过敏反应程度。方法:用SDS-PAGE检测反应条件对产物成分的影响,利用离子交换和分子筛凝胶层析对修饰产物进行分离纯化,通过SDS-PAGE和HPLC检测聚乙二醇化天花粉蛋白(PEG-TCS)的纯度,小鼠引产实验测定PEG-TCS体内生物学活性,用TCS和PEG-TCS作为抗原对豚鼠进行致敏和攻击。结果:在PEG修饰TCS的反应中,PEG与TCS的投料(质量)比和pH值是影响修饰效率的主要因素,修饰反应一般在24～48h内达到平衡;纯化的PEG-TCS经SDS-PAGE检测纯度大于95%,HPLC显示单一峰;小鼠引产实验表明,经过PEG修饰后的TCS,引产活性加强;豚鼠过敏反应实验表明,PEG-TCS具有极低的全身性过敏反应。结论:PEG修饰TCS的最佳反应条件为:TCS与PEG投料比为1:6,pH6．0,反应时间为48h,催化剂氰基硼氢化钠浓度为40mmol·L~(-1);PEG-TCS纯度大于95%; PEG-TCS体内活性能较好的保留,且全身性过敏反应降低。     

抗菌肽生产工艺研究

对解淀粉芽孢杆菌发酵生产抗菌肽的发酵pH、发酵温度和溶氧浓度等条件进行了研究,并通过Box-Benhnken设计及响应面分析法确定了最佳的培养条件.结果表明:该菌株最佳发酵条件为pH值5.8,温度为28℃,溶氧浓度控制在40％左右,发酵液中抗菌肽活性为3604u/mL,较优化前提高了38％.     

酶法合成6-氯嘌呤核苷

利用菌种Lactobacillus helveticus（ATCC 10697）所产生的N-脱氧核糖转移酶,以底物鸟苷和6-氯嘌呤为原料通过碱基交换合成6-氯嘌呤核苷,并优化了反应条件,25 m1的锥形瓶装有反应液10 ml,结果显示最佳反应条件如下：底物最适反应浓度为30 mmol/L（鸟苷）和10 mmol/L（6-氯嘌呤）,菌体添加量8%～10%（湿重）,反应温度40℃,0.1 mol/L磷酸缓冲液（pH值6.0）,摇床转速120 r/min,反应时间24 h,6-氯嘌呤核苷收率可达到51.6%,具有较好的应用前景.     

免疫透射比浊法测定微量白蛋白的研究

目的 对免疫透射比浊法测定微量白蛋白（ALB）进行研究探讨,确定最佳反应条件。方法采用免疫透射比浊法,聚乙二醇（PEG）作为增敏剂,改变PEG浓度,PEG分子量,缓冲液pH值,以及缓冲液体系,对微量白蛋白进行测定。结果采用PEG6000作为增敏剂,PEG浓度为4％,PBS作为缓冲液,pH值为6．5的条件下,浊度最高,检测效果最好,检出限为0．0587μg／ml。检测线性范围为1～8μg／ml,相关系数R=0．9974。结论确定免疫透射比浊法测定微量白蛋白的最佳反应条件,对临床诊断试剂盒的研发具有指导意义。     

响应面法优化花生凝集素修饰长春花碱隐形阳离子脂质体处方

目的:采用响应面法优化花生凝集素(PNA)修饰长春花碱隐形阳离子脂质体的处方。方法:采用硫酸铵梯度法制备花生凝集素修饰长春花碱隐形阳离子脂质体;以半数抑制浓度IC₅₀为指标,考察卵磷脂与3β-[N-(N',N'-二甲基胺乙基)胺基甲酰胺基]胆固醇(EPC/DC-Chol)摩尔比、卵磷脂与聚乙二醇-二硬脂酰磷脂酰乙醇胺(EPC/ PEG₂₀₀₀-DSPE)摩尔比、花生凝集素(PNA)质量百分比3个因素对考察指标的影响,并对各个因素进行二项式拟合,通过响应面Box-Behnken设计优选最佳处方。结果:优选出的处方为EPC/DC-Chol 摩尔比为1.5:1、EPC/ PEG_2000-DSPE摩尔比20:1、PNA质量百分比为0.1%。按照优化后的处方所制备脂质体的Zeta电位稳定、粒径分布均匀。结论:该方法应用简便、预测性好,制备的PNA修饰长春花碱隐形阳离子脂质体符合设计要求。     

星点设计-效应面法优化转铁蛋白修饰粉防己碱与硫酸长春新碱脂质体的处方

目的:制备转铁蛋白(TF)修饰粉防己碱(TET)与硫酸长春新碱(VCR)的主动靶向脂质体,优化其处方。方法:以硫酸铵梯度法制备TF修饰TET与VCR脂质体,以TET包封率、VCR包封率的综合评分为指标,用星点设计-效应面法优化卵磷脂/胆固醇(EPC/Chol)摩尔比、卵磷脂/聚乙二醇2000-二硬脂酰磷脂酰乙醇胺(EPC/PEG2000-DSPE)摩尔比、TF质量分数,并进行验证试验。结果:最优处方为EPC/Chol摩尔比1.5∶1,EPC/PEG2000-DSPE摩尔比20∶1,TF质量分数0.10%;所得脂质体TET包封率为97.80%,VCR包封率为93.00%,综合评分为94.44(n=3),与其预测值93.81接近。结论:优化所得处方稳定,可用于制备TF修饰TET与VCR脂质体。     

响应面分析法优化牡丹皮中总黄酮的提取工艺

目的:采用响应面分析法优化牡丹皮中总黄酮的提取工艺。方法:固定提取时间,以乙醇浓度、料液比、提取温度为响应因子,总黄酮得率为响应值,采用3因素3水平的响应面分析法优化牡丹皮中总黄酮的提取工艺参数。结果:各因素对总黄酮提取率的影响大小依次为料液比>提取温度>乙醇浓度;牡丹皮总黄酮的最佳提取工艺条件为乙醇浓度60%,提取温度55℃,料液比1∶40(W/V),在此条件下,牡丹皮总黄酮得率为3.11mg·g-1。结论:试验结果与模型预测值相符,该工艺可用于牡丹皮中总黄酮的提取。     

Preparation and properties of alginate lyase modified with poly(ethylene glycol)

Modification of the enzyme alginate lyase (AL) with poly(ethylene glycol) (PEG) was attempted for the degradation and removal of alginate biofilms in infectious diseases. The modification of AL with PEG was attempted with three kinds of N-succinimidyl succinate PEG (SS-PEG), which differed in molecular weight (i.e., 2000, 5000 and 12,000 Da). The conjugation of PEG to free amino groups on AL was confirmed by gel permeation chromatography. Quantification of residual free amino groups revealed that PEG modification progressed further with a higher pH and a larger molar ratio of SS-PEG to AL. The reproducibility of the reaction was fairly good. The enzyme activity decreased with increasing PEG modification but the immunoreactivity toward anti-AL antibodies, as evaluated by an ELISA method, was much more remarkably reduced. The immunoreactivity was more reduced by the conjugated PEG with the larger molecular weight. In the reaction with PEG of molecular weight 12,000 Da, we obtained PEG-modified AL retaining approximately 40% enzyme activity but only 0.5% of the immunoreactivity of native AL.     

聚乙二醇对生长激素释放肽的化学修饰

目的为得到生长激素释放肽-2(growth hormone releasing peptide-2,GHRP-2)的聚乙二醇(mPEG)修饰产物并考察其稳定性。方法采用mPEG-NHS活性酯,在不同的溶剂条件下,用不同的投料比对GHRP-2进行修饰;HPLC检测反应结果;Sephadex 75凝胶层析法进行分离纯化。结果确定以无水二甲基甲酰胺为溶剂,m(PEG-NHS)∶m(GHRP-2)=1∶1为最佳条件,原料基本消失,主要得到单修饰的GHRP-2。所得产物在pH7.4、pH8.4、pH9.4缓冲液中50 d时的释药率分别为2.1%、7.2%、20.5%。结论无水条件更有利于mPEG-NHS活性酯对GHRP-2的修饰,产物在体外弱碱性条件下较稳定。     

星点设计-效应面法优化尼可地尔胃漂浮缓释片制剂处方

目的:应用星点设计-效应面法对尼可地尔胃漂浮缓释片进行处方优化。方法:采用粉末直接压片法,以制剂辅料中HPMC、十八醇和碳酸氢钠的用量为考察因素,以1,4,8 h的累积释放度和漂浮性能为评价指标,应用星点设计-效应面法优化制剂处方,并对胃漂浮缓释片释药机理做初步研究。结果:最佳制剂处方为:HPMC 60 mg、十八醇40 mg、碳酸氢钠30 mg;经处方验证,体外累积释放度的预测值与实测值偏差<5%,起漂时间<5 min,续漂时间>8 h;经Ritger-Peppas方程拟合,n=0.5357,提示该缓释片体外释放为非Fick扩散,具扩散与骨架溶蚀的双重机制。结论:应用星点设计-效应面法优选出了最佳制剂处方,按最佳制剂处方制备的尼可地尔胃漂浮缓释片具有良好的漂浮性能和缓释特性,制备方法简便。     

Box-Behnken响应面法优化延胡索配方颗粒的提取工艺

目的优选延胡索配方颗粒中延胡索总生物碱提取的工艺条件。方法采用单因素实验结合Box-Behnken响应面法,以延胡索总生物碱提取率为评价指标进行实验,考察提取时间、水料比和提取次数3个因素对提取工艺的影响。结果根据回归方程确定的最佳提取工艺条件为:提取时间100min;水料比12.5∶1(mL·g-1);提取次数3次。结论采用该工艺条件,延胡索总生物碱的提取率为1.94%,与理论值预测值接近,具有一定的实用价值。     

The molar hydrodynamic volume changes of factor VIIa due to GlycoPEGylation

The effects of GlycoPEGylation on the molar hydrodynamic volume of recombinant human rFVIIa were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10kDa PEG and a branched 40kDa PEG, respectively. Molar hydrodynamic volumes were determined by capillary viscometry and mass spectrometry. The intrinsic viscosities of rFVIIa, its two GlycoPEGylated compounds, and of linear 8kDa, 10kDa, 20kDa and branched 40kDa PEG polymers were determined. The measured intrinsic viscosity of rFVIIa is 6.0mL/g, while the intrinsic viscosities of 10kDa PEG-rFVIIa and 40kDa PEG-rFVIIa are 29.5mL/g and 79.0mL/g, respectively. The intrinsic viscosities of the linear PEG polymers are 20, 22.6 and 41.4mL/g for 8, 10, and 20kDa, respectively, and 61.1mL/g for the branched 40kDa PEG. From the results of the intrinsic viscosity and MALDI-TOF measurements it is evident, that the molar hydrodynamic volume of the conjugated protein is not just an addition of the molar hydrodynamic volume of the PEG and the protein. The molar hydrodynamic volume of the GlycoPEGylated protein is larger than the volume of its composites. These results suggest that both the linear and the branched PEG are not wrapped around the surface of rFVIIa but are chains that are significantly stretched out when attached to the protein.     

Assessment of insulin stability inside diblock copolymer PEG-PLA microspheres

Insulin-loaded PEG2-PLA40 and PEG5-PLA20 microspheres containing 5% bovine insulin were manufactured using single emulsion and w/o/w multiple emulsion-solvent evaporation techniques. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the loading efficiencies were higher in case of insulin loaded PEG2-PLA40 and PEG5-PLA20 microspheres prepared by single emulsion emulsion-solvent evaporation technique. Insulin release was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 6, 12 and 18 days of PEG2-PLA40 microspheres erosion showed that insulin was intact inside the eroding microspheres. In addition, only small amounts of protein undergo degradation under these conditions (only 11.69% ± 1.13 of the initially loaded insulin loading were detected as degradation products after 18 days. Mass spectra recorded at these retention times confirmed the presence of insulin with a molar mass of 5734 Da and other two products of molar masses of 5587 Da and 5487 Da.     

Mathematic Modeling for Optimum Conditions on Aflatoxin B1 Degradation by the Aerobic Bacterium Rhodococcus erythropolis

Response surface methodology was employed to optimize the degradation conditions of AFB₁ by Rhodococcus erythropolis in liquid culture. The most important factors that influence the degradation, as identified by a two-level Plackett-Burman design with six variables, were temperature, pH, liquid volume, inoculum size, agitation speed and incubation time. Central composite design (CCD) and response surface analysis were used to further investigate the interactions between these variables and to optimize the degradation efficiency of R. erythropolis based on a second-order model. The results demonstrated that the optimal parameters were: temperature, 23.2 °C; pH, 7.17; liquid volume, 24.6 mL in 100-mL flask; inoculum size, 10%; agitation speed, 180 rpm; and incubation time, 81.9 h. Under these conditions, the degradation efficiency of R. erythropolis could reach 95.8% in liquid culture, which was increased by about three times as compared to non-optimized conditions. The result by mathematic modeling has great potential for aflatoxin removal in industrial fermentation such as in food processing and ethanol production.