Q Sepharose^TM XL强阴离子交换色谱快速纯化猪心中肌红蛋白和细胞色素C

以Q SepharoseTM XL强阴离子交换色谱结合Sephadex G-75分子排阻色谱快速提取纯化猪心中肌红蛋白（Myoglobin,Mb）和细胞色素C（Cyt C）。在pH值为7.6-8.5范围内,Cyt C吸附在Q SepharoseTMXL强阴离子交换柱上,而Mb会缓慢通过。所以选择QSepharoseTMXL强阴离子交换柱的上样pH值为7.9,洗脱pH值为7.2。电泳检测证明,经过QSepharoseTMXL柱初步纯化所得Mb和Cyt C的产率分别为89.7%和93.2%;再经过Sephadex G-75分子排阻色谱可得到纯的Mb和Cyt C,其产量分别为77.43mg.（kg猪心）-1和86.42mg.（kg猪心）-1。使用QSepha-roseTMXL强阴离子交换色谱能从猪心中快速制备纯的Mb和Cyt C。     

Q SepharoseTM XL强阴离子交换色谱快速纯化猪心中肌红蛋白和细胞色素C

以Q SepharoseTM XL强阴离子交换色谱结合Sephadex G-75分子排阻色谱快速提取纯化猪心中肌红蛋白（Myoglobin,Mb）和细胞色素C（Cyt C）。在pH值为7.6-8.5范围内,Cyt C吸附在Q SepharoseTMXL强阴离子交换柱上,而Mb会缓慢通过。所以选择QSepharoseTMXL强阴离子交换柱的上样pH值为7.9,洗脱pH值为7.2。电泳检测证明,经过QSepharoseTMXL柱初步纯化所得Mb和Cyt C的产率分别为89.7%和93.2%;再经过Sephadex G-75分子排阻色谱可得到纯的Mb和Cyt C,其产量分别为77.43mg.（kg猪心）-1和86.42mg.（kg猪心）-1。使用QSepha-roseTMXL强阴离子交换色谱能从猪心中快速制备纯的Mb和Cyt C。     

精氨酸激酶C端结构域的克隆及其表达纯化

精氨酸激酶(Arginine kinase,AK)由一个小的球形N端结构域和一个大的C端结构域构成。将来源于基围虾的AK C端结构域基因成功克隆到了原核表达载体pET-28a上,然后再转入Rosetta进行表达。探讨了AK C端结构域的最佳表达条件,当OD600达到0.5~0.7时,用终浓度为0.2 mmol.L-1的异丙基硫代--βD-半乳糖苷(IPTG)在20℃下诱导培养5~6 h,AK C端结构域在上清中大量表达。采用His-Tag金属螯合亲和层析纯化、不连续梯度咪唑洗脱,得到了电泳纯的AK C端结构域。     

肺炎链球菌表面蛋白A的原核表达、纯化及其多克隆抗体的制备

目的原核表达、纯化肺炎链球菌表面蛋白A(pneumococcal surface protein A,Psp A),并制备多克隆抗体。方法应用ANTHEWIN、DNAstar等分子生物学软件,对Psp A氨基酸序列进行分析,筛选出抗原表位富集区(第33~109个氨基酸),选用原核生物偏爱的密码子优化基因序列,化学合成全新的基因序列pspa,插入质粒p GEX-4T-2和p ET28a(+)中,构建重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa,转化大肠埃希菌BL21(DE3),IPTG诱导表达。分别纯化带有GST标签和His标签的Psp A重组蛋白GST-Psp A和His-Psp A,以His-Psp A作为免疫原,经背部多点免疫新西兰大耳白兔,间接ELISA法检测血清抗体效价,Western blot法检测血清抗体特异性。结果两种重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa经双酶切鉴定构建正确;表达的两种重组蛋白GST-Psp A和His-Psp A相对分子质量分别约为33 000和18 000,均为可溶性表达,纯化后目的蛋白条带均无降解,纯度约为95%,蛋白浓度分别为2和0.2 mg/ml;制备的兔抗血清效价较高,可达1∶200 000,且特异性较好。结论原核表达并纯化了肺炎链球菌Psp A融合蛋白,并制备了特异性良好的高效价兔抗血清,为下一步建立肺炎链球菌快速检测技术奠定了基础。     

强碱性阴离子交换树脂吸附分离铂族元素条件的探讨

采用强碱性阴离子交换树脂吸附分离贵金属铂族元素,并对吸附分离条件酸性、吸附容量、流速、树脂粒度、溶液放置时间及Rh在不同配合物存在下的吸附情况进行了研究和探讨。     

201×4强碱性阴离子交换树脂吸附Cr(Ⅵ)的机理研究

实验研究了201×4强碱性阴离子交换树脂去除溶液中Cr(VI)的影响因素以及该树脂吸附Cr(VI)的动力学和热力学特性。结果表明,吸附平衡过程符合Frendilch等温吸附方程,吸附动力学数据表明化学反应过程为控速步骤,树脂对Cr(VI)的吸附量随pH的降低而增加,随着温度的升高而增加,表观吸附活化能Ea(10mg/L)=30.851kJ-mol-1,Ea(400mg/L)=16.074kJ·mol-1。当pH=2.0时,反应温度为323K,吸附量存在最大值为13.74mg/g。树脂的再生能力比较强,经过3次再生,树脂的平衡吸附量下降8.3%。     

MDCK宿主细胞残留蛋白多克隆抗体的制备、纯化及初步应用

目的 制备MDCK宿主细胞残留蛋白多克隆抗体,并进行纯化及初步应用,为进一步研制宿主细胞残留蛋白检测试剂盒奠定基础.方法 无血清悬浮培养MDCK细胞,采用不同方法(反复冻融、裂解液、超声)裂解细胞获得全细胞蛋白抗原.将抗原与弗氏佐剂乳化后经背部多点皮下免疫家兔,间接ELISA法检测血清抗体效价,当抗体效价≥1 × 10...     

甲氰菊酯多克隆抗体的制备

利用水溶性碳二亚胺法将甲氰菊酯的合成前体甲氰菊酸与牛血清蛋白偶联,合成了甲氰菊酯的免疫半抗原,免疫新西兰大自兔获得了甲氰菊酯多克隆抗体.通过紫外扫描分析,免疫原中甲氰菊酸分子与蛋白质分了的偶联比为8.8:1,50%饱和硫酸铵法纯化抗体,间接ELISA法测定多克降抗体效价达1:12 800,最适甲氰菊酸-OVA包被抗原质量浓度为1.0 mg/L;最低检出限为8.5μg/L.以抗体与甲氰菊酯的反应率为100%,抗体与其他4种菊酯类农药交叉反应率很低,与溴氰菊酯交叉反应较高.结果表明:制备的甲氰菊酯多克隆抗体具有较高的特异性,可用于甲氰菊酯残留的检测.     

特异性抗cTnI多克隆抗体的制备

目的 制备特异性抗人心肌肌钙蛋白1（cTnI）多克隆抗体。方法 用人cTnI免疫家兔,制备抗人cTnI多克隆抗体,采用膜渗滤亲和层析法吸附掉与骨骼肌肌钙蛋白1（aTnI）有交叉反应的多抗,制备特异性抗cTnI多克隆抗体。结果 抗体经槽式对流兔疫电泳、酶联免疫吸附试验证明对cTnI具有高度特异性,与sTnI无交叉反应。结论 为应用一步夹心酶联免疫吸附法检测cTnI奠定了基础。     

阴离子交换层析法纯化聚唾液酸工艺

以大肠杆菌发酵所产聚唾液酸粗品(含蛋白质、核酸、无机盐和色素等杂质)为纯化对象,通过静态吸附和梯度洗脱实验选择了分离效果较好的阴离子交换介质Q-琼脂糖凝胶FF,对聚唾液酸的纯化工艺条件进行优化. 得到最佳洗脱条件为：洗脱液为pH 7.2的NaCl-0.02 mol/L磷酸钠缓冲液体系,流速0.8 mL/min,洗脱线性梯度方程为CNaCl=0.0017VEluent (CNaCl为NaCl浓度,VEluent为洗脱液体积),层析柱体积46 mL时最大进样量4.0 mL. 该条件下聚唾液酸回收率在86.0%以上,纯化后样品中的蛋白质含量从1.9%降低至0.04%,纯度在98%以上. 紫外吸收光谱和高效凝胶过滤色谱分析表明,聚唾液酸产品组分均一,重均分子量为303 kDa.     

脱氧雪腐镰刀菌烯醇的快速分离纯化及其抗体制备

采用高速逆流色谱法结合高效液相色谱法从禾谷镰刀菌固体发酵产物中分离纯化脱氧雪腐镰刀菌烯醇(DON)。先用高速逆流色谱在以乙酸乙酯-水(1∶1,体积比)为两相溶剂系统、温度为25℃、主机转速为890r·min-1、流速为1.0mL·min-1、检测波长为254nm的条件下进行分离,再用高效液相色谱进一步分离纯化,最终从300g大米发酵培养物中得到了8mg DON,纯度为97.7%。将纯化的DON用于DON人工抗原制备,基质辅助激光解吸附电离串联飞行时间质谱分析表明,人工抗原制备成功;免疫动物后,研制出了高效价的多克隆抗体。本研究建立的色谱方法操作简单,分离效果较好,纯化的DON可以用于抗体的制备。     

Separation of Gallium and Indium Isotopes by Cation and Anion Exchange Chromatography

Ion exchange chromatography was applied to study chemical isotope effects of gallium and indium in ligand exchange reactions. A strongly acidic cation and a strongly basic anion exchange resin were used as a solid phase, and aqueous HCl as a liquid phase. On the cation exchanger, the light isotope ⁶⁹Ga was enriched at the front part of the elution band and the heavy isotope ⁷¹Ga at the end part. Instead, the light ¹¹³In isotope was enriched at the end part, and the heavy isotope ¹¹⁵In at the front part. The isotope separation factor ? is equal to 3.3?×?10^–5 for gallium and 2.0?×?10^–4 for indium. On the anion exchanger, the heavy gallium isotope was enriched at the front part, whereas the heavy indium isotope at the end part of the band, with ? equal to ～10^–3 and 1.7?×?10^–4, respectively. This pattern of enrichment is caused by stronger Ga³⁺–OH₂ than Ga³⁺–Cl^? bond, and by inverse order of bond strength for indium. In the displacement method, gallium and indium on anion exchanger also show opposite enrichment of their isotopes, but the ? values (1.5?×?10^–2 for gallium and 5?×?10^–3 for indium) are greater than those found in the elution method, probably due to much higher concentrations of the metals.     

Mixed-Beds of Strong and Weak Anion Exchange Resins for Protein Separations with Step-Induced pH Gradients

A new way of performing pH gradient-based protein separations using a mixed-bed column comprising a small-pore weak base resin and a large-pore strong base resin and a step change in pH at the column entrance is introduced. The approach effectively decouples intracolumn generation of pH gradients, which ensues from ionic interactions with the weak base resin, from protein separation, which ensues from interactions with the strong base resin ligands, resulting in a process that is more easily predictable, less prone to the effects of protein loading on the pH gradient, less susceptible to hydrophobic interactions between protein and resin surface, and more flexible. The approach is demonstrated successfully using a tertiary amine resin as the weak base component together with a range of commercial strong anion exchangers with quaternary amine functionality. A predictive model, based on the potentiometric titration of the weak base resin, is in excellent agreement with experimentally determined pH profiles obtained using mixed-bed columns and amine buffers with pH steps from 9 to 7. The separation of protein charge variants with the mixed-bed concept is demonstrated using these columns for a deamidated monoclonal antibody mixture, which yielded highly homogeneous fractions.     

反相色谱法研究人粒细胞集落刺激因子的离子交换色谱复性和稀释复性

以反相色谱分析为主要手段,结合活性测定,研究了以包含体形式表达的重组人粒细胞集落刺激因子(rhG-CSF)的稀释复性和离子交换色谱复性. 建立了L-精氨酸离子交换复性蛋白质的方法,将变性、还原的rhG-CSF吸附到高浓度变性剂平衡的离子交换色谱柱上,用L-精氨酸作洗脱剂进行梯度洗脱并同时脱除变性剂,实现了rhG-CSF的复性. 与稀释复性相比,色谱复性的rhG-CSF处于一种中间状态,呈现快速而不同步的动力学特点,这与色谱复性的梯度洗脱及固定相的吸附有关. 同时发现了对复性峰进一步稀释并且温育则可以提高其活性的新现象.     

用离子交换法分离豆饼中异黄酮的研究

在pH=10.5的碱性介质中,大豆异黄酮被强碱阴离子交换树脂交换吸附,用2.5×10-2mol/LHCl溶液定量洗脱。用80%乙醇溶液提取豆饼中大豆异黄酮的粗提物,经离子交换分离,可使产品中大豆异黄酮含量提高2.43~10.57倍,回收率达96%。     

Optimal Integration of Directly Combined Hydrophobic Interaction and Ion Exchange Chromatography Purification Processes

Hydrophobic interaction and ion exchange chromatography are basic steps in purification of fermentative biopharmaceuticals. An optimization by statistical design of experiments requires a huge amount of feed. An alternative approach is the combination of model parameter determination using small scale experimental model parameter determination (1‐mL columns) and rigorous process modeling. Applicability for the prediction of the separation of a fermentation mixture of CHO mammalian cell culture is validated and hence IgG is purified from cell culture supernatant. Hydrophobic interaction chromatography directly combined with ion exchange chromatography is optimized. Any direct integration of those two main unit operations in purification processes is a methodological first step towards total process optimization. The potential for cost reduction and overall yield improvement is demonstrated and this leads to the conclusion that single step optimization is a feigned and not a real optimum.     

Predicting Retention and Resolution of Protein Charge Variants in Mixed-Beds of Strong and Weak Anion Exchange Resins with Step-Induced pH Gradients

A model is developed to predict protein retention and resolution in mixed-bed columns containing a mixture of a small-pore weak base resin and a large-pore strong base resin. Using amine buffers, the weak base resin generates smooth pH gradients when the column is subjected to pH steps, while the strong base resin interacts with the proteins effecting the separation. The model describes simultaneously ion exchange interaction on the weak base resin and protein adsorption and transport on the strong base resin. The separation of a monoclonal antibody (mAb) from a charge variant whose pI is about 0.2 pH units lower is studied experimentally with the anion exchange resins AG 4-X4 and SOURCE 30Q as the weak and strong base components, respectively. The properties of each resin are characterized independently and model parameters are obtained experimentally for each. Model predictions of pH gradient-based separations in columns containing different proportions of the two resins and at different buffer concentrations are found to be in agreement with experimental results. The model can thus be used as a process design tool to predict combinations of buffer and mixed-bed compositions, column lengths, and mobile phase velocities that would yield a desired separation performance.     

阴离子交换树脂对茶氨酸的吸附动力学和热力学

以酶法制备茶氨酸,研究了碱性条件下阴离子交换树脂对茶氨酸的吸附与分离. 结果表明,强碱性树脂对茶氨酸的吸附性能优于弱碱性树脂,且其吸附容量受pH值的影响较小,pH=9.0时凝胶型强碱性树脂HZ202对茶氨酸的平衡吸附量可达96.3 mg/g. 对HZ202吸附茶氨酸的吸附等温模型及动力学、热力学参数进行了分析,结果表明,茶氨酸在HZ202树脂表面为非均一分布,Spis模型可较好模拟其吸附等温线数据;热力学参数计算结果显示,不同温度下吸附过程的吉布斯自由能变DG均为负值,表明吸附为自发的放热过程;吸附过程的焓变DH=20.9~418.4 kJ/mol,可判断其为化学吸附. 茶氨酸吸附过程符合准二级动力学方程,吸附过程受化学反应控制,提高茶氨酸初始浓度可提高吸附速率.