百日咳丝状血凝素酶联免疫检测法的建立

目的建立百日咳组分疫苗丝状血凝素(FHA)抗原含量监控的ELISA检测法。方法制备的多克隆抗血清,经辛酸硫酸铵法纯化抗体,用过碘酸钠氧化法辣根过氧化物酶标记,以棋盘滴定法确定最佳包被抗体及酶标抗体的浓度配伍,建立了双抗体夹心ELISA检测法。结果对双抗体夹心ELISA法的特异性、最佳线性范围、检测限度、精密度、准确度、测定限量、适用性的一系列验证试验表明,该方法与百日咳组分疫苗中PT和Prn无明显交叉反应,特异性较好。在0至20 ng/mL测量区间有最佳线性,相关系数大于0.99;经实验内12次及不同试验间3次测定16、8、4 ng/mL中的FHA含量,变异系数在0.2%～11.4%间,回收率在96.9%～114.5%间,精密度及准确度验证均符合常规质控要求,因此测定限量为4 ng/mL。结论该方法能有效检测出百日咳杆菌培养上清中的FHA含量,可用于百日咳组分疫苗生产过程的中间品质量控制。     

国产层析填料在百日咳毒素和丝状血凝素纯化中的筛选及应用

目的 筛选适合的国产层析填料以满足对组分百日咳疫苗中百日咳毒素(pertussis toxin, PT)与丝状血凝素(filamentous hemagglutinin, FHA)的分离纯化。方法 筛选对PT与FHA结合能力较好且分离度高的国产层析填料，优化纯化工艺，通过3批纯化试验比较国产填料与进口填料的抗原回收率、目的抗原纯度以及对目的抗原的动态结合载量。结果 通过纯化试验筛选到国产填料SP resin-1与MMC resin-1并优化了纯化工艺。3批纯化试验显示，SP resin-1纯化百日咳抗原工艺稳定，FHA的纯度和回收率与Capto SP ImpRes差异无统计学意义(P>0.05);且与Capto SP ImpRes相比，SP resin-1对抗原的动态结合载量更高。3批PT精纯试验显示，国产填料MMC resin-1精纯PT的工艺稳定，PT的纯度和回收率均与Capto MMC差异无统计学意义(P>0.05),并且MMC resin-1对PT的载量高于Capto MMC。结论 试验筛选出纯化PT与FHA的国产填料SP resin-1与MMC resin-1,纯...     

百日咳丝状血凝素的纯化及初步鉴定

目的从百日咳鲍特菌中提纯百日咳丝状血凝素(filamentous hemagglutinin,FHA),并对其进行初步鉴定。方法采用珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法,从百日咳鲍特菌培养上清中纯化FHA;SDSPAGE电泳和PAGE电泳分析样品纯度,并经斑点免疫印迹法对其进行鉴定。结果纯化样品的纯度达到95%以上,斑点免疫印迹中和FHA单抗有反应斑点,与百日咳毒素(pertussis toxin,PT)单抗无反应斑点。结论珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法可作为一种快速有效的纯化手段提取纯度较高的FHA,有望作为参考品用于百日咳FHA抗原含量检测、抗FHA血清抗体滴度检测及抗原纯度检测。     

百日咳毒素和丝状血凝素的纯制和检测现况

介绍了百日咳毒素和丝状血凝素的分离纯化及纯度、活性检定的方法,对影响分离纯化这两种活性物质的因素也作了一些分析     

百日咳杆菌丝状血凝素的纯制及其生物学特性的研究

介绍了两种纯制百日咳杆菌丝状血凝素的方法，并对不同方法纯化的丝状血凝素的产量、纯度、生物学活性等方面进行了比较，从中选择出一种纯度高、活性好、可以大批量提纯的方法。     

溶液环境对百日咳疫苗分离纯化的影响

针对百日咳疫苗在低盐条件下不稳定, 容易聚集而导致层析过程收率低、分离度低的难题, 实验中选择脲作为稳定剂来改善百日咳疫苗所处的溶液环境, 并采用离子交换层析和凝胶过滤层析进行百日咳疫苗的分离纯化, 通过ELISA抗原活性测定和还原性SDS-PAGE等方法研究了脲对百日咳疫苗分离纯化的影响。结果表明, 在流动相中加入 2 mol/L脲作为稳定剂, 能显著提高离子交换层析和凝胶过滤层析中的PT和FHA活性回收率、凝胶过滤层析的分离度、PT和FHA的纯度。这些结果对百日咳疫苗的分离纯化和层析工艺优化提供了重要的依据和参考。     

百日咳杆菌去内毒素及保护性抗原的纯化

内毒素(Endotoxin,简称ET)是百日咳全菌苗(Bordetellapertussis vaccine)产生副作用的主要毒素之一,且不易除去。现有的分离方法,如蔗糖密度梯度离心法,较繁琐,成本高。本文采用Sepha-cryl S-300凝胶层折法可以简便有效的去除大部分内毒素。初步毒素试验结果表明:已达到日本生物制品规格的要求。两种保护性抗原FHA和LPF-HA也得到进一步分离纯化,为今后研制高效的百日咳组分菌苗提供了实验条件。     

响应面法优化康定鼠尾草中迷迭香酸提取工艺

利用单因素实验和响应面法优化康定鼠尾草中迷迭香酸提取工艺。采用高效液相色谱法测定迷迭香酸含量,以提取率为参考指标。通过单因素实验筛选出料液比、提取时间和乙醇浓度三个主要因素,通过BoxBehnken设计方案,建立迷迭香酸提取得率的二次回归方程,得到优化组合条件。最佳提取条件为乙醇浓度40%,液固比15∶1,提取时间50 min。最佳提取条件下,迷迭香酸提取得率为12.30 mg/g。优化得到的康定鼠尾草中迷迭香酸的提取工艺合理,操作可行,质量稳定。     

响应面法优化超声提取绿茶茶多酚工艺

利用响应面法对超声提取绿茶荼多酚的工艺条件进行优化,在单因素试验的基础上,根据中心组合设计原理采用三因素三水平的响应面分析法,依据回归分析确定最优提取工艺条件。结果表明,其最佳工艺条件为：液料比为40．2mL／g,超声功率为476W,提取时间为15．1min,采用该工艺条件,茶多酚的提取得率达到10．312％,通过响应面法得到一个能较好预测试验结果的模型方程。     

响应面法优化白头翁总黄酮提取工艺

采用无水乙醇C2H5OH/硫酸铵(NH4)2SO4双水相体系分离白头翁中的黄酮。确定双水相体系组成为21%C2H5OH/22%(NH4)2SO4,通过单因素试验和Box-Benhnken实验设计探讨黄酮粗提液质量分数、NaCl质量分数和pH值对萃取效果的影响。确定最佳萃取条件为:黄酮粗提液质量分数12.5%,NaCl质量分数1.5%,pH 5.99,在此条件下,白头翁总黄酮主要分布在上相,萃取率可达73.6%。     

响应面法优化南方山荷叶中鬼臼毒素的提取工艺

鬼臼毒素（Podophyllotoxin）是一种常用的临床天然药物。本研究以鬼臼类药材南方山荷叶（Diphylleia sinensis H.L.Li）为材料,以提取物中鬼臼毒素的含量为控制指标,应用响应面法优化南方山荷叶中鬼臼毒素的提取工艺。采用高效液相色谱法测定提取物中鬼臼毒素的含量,在单因素实验筛选的基础上,选择出对鬼臼毒素提取率影响较大的3个因素：乙醇浓度、提取温度和提取时间,然后利用Design-expert软件设计3因素3水平响应面实验,最终获得鬼臼毒素提取率最高时的提取条件为：乙醇浓度48.18%、提取温度62.66℃、提取时间16.15 h,此时鬼臼毒素的理论提取率可达7.636%。进一步对该条件进行实验验证,3次实验平均值为7.54%,与理论值差异较小,表明该模型拟合良好,实验方法可靠。     

响应面法优化粉被虫草中虫草素的提取工艺

【背景】粉被虫草是民间常用药用真菌,具有抗辐射等多种药理作用,虫草素是其重要的活性物质。【目的】探究粉被虫草中提取虫草素的影响因素,为制取虫草素提供参考。【方法】以虫草素得率为指标,用响应面法优化超声提取工艺。分别考察超声时间、液料比和超声功率对提取效率的影响,在单因素实验基础上进行3因素3水平的响应面分析实验。【结果】最优提取条件为超声时间606 s、液料比45.9:1、超声功率400 W,在此条件下虫草素得率为6.136 mg/g,与模型预测值接近,方程拟合良好。【结论】首次对粉被虫草中虫草素的提取条件进行了研究,将对制取虫草素提供参考,有利于粉被虫草的深度开发利用。     

Isolation and characterization of Aspergillus sp. for the production of extracellular polysaccharides by response surface methodology

In this study, Aspergillus sp. was isolated for the production of extracellular polysaccharide. The process parameters were initially optimized by traditional methods. The cheap substrate, wheat bran was used for the production of extracellular polysaccharide in solid state fermentation. Supplementation of (1%, w/w) maltose, gelatin enhanced EPS production (5.36?mg/g). The salts such as, Cu²⁺ (4.9?mg/g), Ca²⁺ (3.5?mg/g), Zn²⁺ (2.9?mg/g), Mn²⁺ (3.4?mg/g) and Mg²⁺ (1.8?mg/g) stimulated EPS production. In two level full factorial experimental designs, the EPS yield varied from 3.18 to 11.65?mg/g wheat bran substrate with various combinations of the components supplemented with wheat bran substrate. Among these selected factors in central composite design, maltose significantly influenced on extracellular polysaccharide production.     

Application of reverse micelle extraction process for amylase recovery using response surface methodology

The effect of different process variables of reverse micelle extraction process like pH, addition of surfactant (AOT) concentration and potassium chloride (KCl) concentration on amylase recovery has been studied and analysed. Solid-state fermentation was used for the production of amylase enzyme. Response surface methodology (RSM) using central composite rotatable design (CCRD) was employed to analyse and optimize the enzyme extraction process. The regression analysis indicates that the effect of AOT concentration, and KCl concentration were significant, whereas the effect of pH was non-significant on enzyme recovery. For the maximum recovery of enzyme, the optimum operating condition for pH, AOT concentration (M) and KCl concentration were 10.43, 0.05 and 1.00, respectively. Under these optimal conditions, the enzyme recovery was 83.16%.     

Optimization of lipase-catalyzed synthesis of acetylated EGCG by response surface methodology

(−)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives, which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications, were prepared by lipase catalyzed acylation of EGCG with vinyl acetate. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (6–10 h), temperature (30–50 °C), enzyme amount (1.5–2.5% (w/w) of substrate), and substrate molar ratio of EGCG to vinyl acetate (0.5–1.5) on conversion of EGCG. By using multiple regression analysis, the experimental data were fitted to a second order polynomial model. The most suitable combination of variables was 40 °C, 2.12%, 10 h and 1.13 for the reaction temperature, the enzyme amount, the reaction time, and EGCG/vinyl acetate mole ratio, respectively. At these optimal conditions, the conversion yield reached 87.37%. The presence of mono-, di- and tri-acetylated derivatives in acetylated EGCG was confirmed by LC–MS-MS and identified as 5″-O-acetyl-EGCG, 3″, 5″-2-O-acetyl-EGCG and 5′, 3″, 5″-3-O-acetyl-EGCG by NMR.     

响应面法优化飞龙掌血中总生物碱的提取工艺

以飞龙掌血（Toddalia asiatica（L.）Lam.）的根为实验材料,以总生物碱的含量为检测指标,应用响应面法对飞龙掌血中总生物碱的提取工艺进行了优化。结果显示,当提取条件为乙醇体积分数74.76%、料液比20∶1和提取时间32.87 min时,飞龙掌血提取物中总生物碱的理论含量值可达11.09 mg/g,总生物碱含量达到最高值。按照最佳提取工艺条件进行验证,3次平行实验的均值为（10.74 ±0.18）mg/g,与理论值差异较小,表明利用响应面法来优化飞龙掌血中总生物碱的最佳提取工艺模型拟合良好,具有一定实用价值。     

Optimization of extraction process by response surface methodology and preliminary structural analysis of polysaccharides from defatted peanut (Arachis hypogaea) cakes

In this work, response surface methodology was used to determine optimum conditions for extraction of polysaccharides from defatted peanut cake. A central composite design including independent variables, such as extraction temperature (x₁), extraction time (x₂), and ethanol concentration (x₃) was used. Selected response which evaluates the extraction process was polysaccharide yield, and the second-order model obtained for polysaccharide yield revealed coefficient of determination of 97.81%. The independent variable with the largest effect on response was ethanol concentration (x₃). The optimum extraction conditions were found to be extraction temperature 48.7 °C, extraction time 1.52 h, and ethanol concentration of 61.9% (v/v), respectively. Under these conditions, the extraction efficiency of polysaccharide can increase to 25.89%. The results of structural analysis showed that the main composition of defatted peanut cake polysaccharide was α-galactose.     

响应面分析法优化黑根霉胞外多糖发酵工艺

实验以商品化的马铃薯葡萄糖液体培养基为基础培养基,以胞外粗多糖产量为考察指标,运用响应面分析法考察玉米浆浓度、KH₂PO₄浓度和发酵时间3个因素对胞外多糖发酵产量的影响,以获得黑根霉胞外多糖发酵最优工艺,建立高产、稳定、可控的胞外多糖发酵生产工艺技术方案。经响应面分析,各因素按照对响应值的影响顺序为：玉米浆浓度>发酵时间> KH₂PO₄浓度,且玉米浆浓度、发酵时间对胞外多糖产量的影响极显著,KH₂PO₄浓度对胞外多糖产量的影响不显著。胞外多糖发酵最优工艺为：玉米浆3.2mg/mL、KH₂PO₄ 1.5mg/mL和发酵时间132h,在此条件下胞外多糖的最大预测产量为0.824mg/mL。实验重复性好,是一个高产、稳定、可控的胞外多糖发酵生产工艺技术方案,可以指导黑根霉胞外多糖发酵。     

响应面分析法优化耐高温假黄色单胞菌硫氧化性能

【目的】采用响应面分析法对耐高温假黄色单胞菌的硫氧化性能进行优化。【方法】利用Plackett-Burman试验筛选影响菌株硫氧化性能的关键因子。通过最陡爬坡试验逼近最佳值区域,确定响应面试验中心轴,利用Box-Behnken设计和响应面分析法获得关键因素的最佳浓度。采用经典的改良硫酸钡比浊法测定硫酸根含量。【结果】牛肉膏、麦芽糖、镁离子(Mg~(2+))3个因素是影响菌株硫氧化性能的关键因素。响应面分析表明牛肉膏和镁离子的交互作用对硫酸根转化率的影响最大,优化后的结果为:麦芽糖(%)=0.07,牛肉膏(%)=0.11,Mg~(2+)(%)=0.04时,模型有最大值。模型的F值为52.60(P0.000 1),相关系数R2=0.980 2,说明该二次方程是显著的,该模型在整个回归区域内的拟合较好。经模拟堆肥试验验证该菌株可以有效增加堆肥中硫酸根含量。【结论】该模型可用于分析和预测耐高温假黄色单胞菌优化培养基配方,经优化后该菌株硫氧化性能大幅提升,硫酸根转化率由36.89%提高到80%以上,加入堆肥后与基础发酵条件下菌液相比更加有效增加了堆肥中SO_4~(2-)的含量,具有较好的应用前景。     

响应面法优化嗜热链球菌AR333电转化条件

【背景】嗜热链球菌AR333是本实验室从发酵乳中筛选出的一株高产活性胞外多糖乳酸菌。【目的】建立嗜热链球菌AR333高效电转化体系。【方法】通过单因素试验和Box-Behnken响应面法优化电转化条件。【结果】嗜热链球菌AR333最优电转化条件为甘氨酸浓度8.3g/L,OD_(600)为0.8,10%甘油(体积比)和0.5 mol/L蔗糖的电转缓冲液,pIB184质粒80 ng,电场强度14 kV/cm,0.4 mol/L山梨醇、2 mmol/L CaCl_2和20 mmol/L MgCl_2的LM17复苏培养基,复苏时间5 h。【结论】在最优电转化条件下,嗜热链球菌AR333电转化效率达到3.68×10~5 CFU/μg-DNA,比优化前提高了14倍,实现了嗜热链球菌AR333的高效遗传转化,为其功能解析和基因工程改造奠定基础。