利用 6mol LHCl沉淀枯草芽孢杆菌B2菌株的去细胞培养液 ,甲醇抽提获得脂肽类抗生素粗提物 ,过SephadexLH 2 0层析柱获得粗纯化物 ,经MALDI TOF MS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLCSMARTSYSTEM ,将粗纯化物过 ...利用 6mol LHCl沉淀枯草芽孢杆菌B2菌株的去细胞培养液 ,甲醇抽提获得脂肽类抗生素粗提物 ,过SephadexLH 2 0层析柱获得粗纯化物 ,经MALDI TOF MS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLCSMARTSYSTEM ,将粗纯化物过 μRPCC2 C1 8层析柱对表面活性素变异体进行分离后获得纯化物。经MALDI TOF PSD MS对纯化物的结构分析表明 ,B2菌株的表面活性素变异体由 1 3、1 4和 1 5个碳原子的脂肪酸链以及L Glu L Leu D Leu L Val L Asp D Leu L展开更多
A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction...A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The whole procedure can be performed in four hours. Using this purification method, we can reproducibly obtain, from 4 × 109 of proviral cells cultured in roller bottles, purified rAAV-GFP stocks with titers of around 5×1013 particles/mL and purity greater than 95%. The infectious titers of the vector stocks were up to 2×1012 TU/mL, thus particle-to-infectivity rate was about 25. Under an electronic microscope, most rAAV particles appeared full and a few were in intermediate form. Empty particles were rarely seen. The purified rAAV-GFP stocks have been successfully used in in vitro and in vivo transfection experiments. Therefore, this new method offers a simple, rapid and cost-effective way for large-scale rAAV purification.展开更多
前期杂交优化后赤芝菌种经液体深层发酵后,提取灵芝菌丝体多糖,并过DEAE-Sepharose Fast Flow柱分离纯化,利用高效体积排阻色谱(HPSEC)检测多糖级分的纯度,采用完全酸水解PMP柱前衍生化RP—HPLC测定多糖级分的单糖组成,多角度光散射仪...前期杂交优化后赤芝菌种经液体深层发酵后,提取灵芝菌丝体多糖,并过DEAE-Sepharose Fast Flow柱分离纯化,利用高效体积排阻色谱(HPSEC)检测多糖级分的纯度,采用完全酸水解PMP柱前衍生化RP—HPLC测定多糖级分的单糖组成,多角度光散射仪联用装置(SEC—MALLS)测定其绝对重均分子量(Mw),并且根据分子旋转半径与分子摩尔数的关系曲线斜率初步推断其空间构象。结果显示:分离纯化得到3个多糖级分GLMP1、GLMP2和GLMP3,HPSEC检测其峰面积百分比分别为93.58%,97.64%,99.19%,单糖组成分析结果表明GLMP1、GLMP2和GLMP3均含有甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖和岩藻糖,但单糖摩尔比各异。SEC—MALLS测试GLMP1、GLMP2和GLMP3的Mw分别为4.526×105,4.603×104,3.760×103 g/mol,3个多糖级分构象可能均为高度紧缩且具有分支结构的聚合物。展开更多
通过采用匀浆、硫酸铵盐析、透析、DEAE-52阴离子交换层析、Sephacryl S-300凝胶层析等方法从海参体壁中分离纯化并得到了α-1,4淀粉酶。结果表明,该酶具有3条亚基链,分子质量约为420 k D;最适p H值为9.0,最适温度为80℃,90℃条件下保温...通过采用匀浆、硫酸铵盐析、透析、DEAE-52阴离子交换层析、Sephacryl S-300凝胶层析等方法从海参体壁中分离纯化并得到了α-1,4淀粉酶。结果表明,该酶具有3条亚基链,分子质量约为420 k D;最适p H值为9.0,最适温度为80℃,90℃条件下保温30 min后仍有38%以上的相对酶活性,因此,该酶是一种具有较高热稳定性的碱性高温淀粉酶。K+、Fe3+和Mn2+对其有较强的抑制作用,Cu2+和Ca2+对其有较强的激活作用。展开更多
采用Sepharose CL-6B凝胶柱纯化壶瓶枣多糖(polysaccharides from Zizyphus jujube Mill.cv.Hupingzao,简称ZJP)ZJP-2和ZJP-5组分,并对纯化后多糖的结构进行分析。结果表明:经纯化后得到ZJP-2b和ZJP-5a两种组分均一的壶瓶枣活性多糖,分...采用Sepharose CL-6B凝胶柱纯化壶瓶枣多糖(polysaccharides from Zizyphus jujube Mill.cv.Hupingzao,简称ZJP)ZJP-2和ZJP-5组分,并对纯化后多糖的结构进行分析。结果表明:经纯化后得到ZJP-2b和ZJP-5a两种组分均一的壶瓶枣活性多糖,分子质量分别为89.21、61.60 k D,均具备多糖的特征吸收峰,且均以β-构型的吡喃糖为主;ZJP-2b中单糖组成主要为鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖,其物质的量比为32.4∶9.5∶9.4∶14.7∶9.7,而ZJP-5a中单糖组成主要为鼠李糖、阿拉伯糖、木糖、甘露糖和半乳糖,其物质的量比为20.2∶42.9∶2.2∶7.5∶14.5;当质量浓度为3.5 mg/mL时,ZJP-2b和ZJP-5a的羟自由基清除率分别为30.51%和57.22%。展开更多
A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43...A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion ex- change chromatography on DEAE-Sephadex As0. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60~C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low Ki value of 0.2 ~tM. The inhibitoi- was found to be susceptible to varying concentrations of reducing agents like DTT and 2- mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth ofPieris brassi- cae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.展开更多
文摘利用 6mol LHCl沉淀枯草芽孢杆菌B2菌株的去细胞培养液 ,甲醇抽提获得脂肽类抗生素粗提物 ,过SephadexLH 2 0层析柱获得粗纯化物 ,经MALDI TOF MS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLCSMARTSYSTEM ,将粗纯化物过 μRPCC2 C1 8层析柱对表面活性素变异体进行分离后获得纯化物。经MALDI TOF PSD MS对纯化物的结构分析表明 ,B2菌株的表面活性素变异体由 1 3、1 4和 1 5个碳原子的脂肪酸链以及L Glu L Leu D Leu L Val L Asp D Leu L
基金the National High-technology Development Program (Grant No. Z20-05-02).
文摘A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The whole procedure can be performed in four hours. Using this purification method, we can reproducibly obtain, from 4 × 109 of proviral cells cultured in roller bottles, purified rAAV-GFP stocks with titers of around 5×1013 particles/mL and purity greater than 95%. The infectious titers of the vector stocks were up to 2×1012 TU/mL, thus particle-to-infectivity rate was about 25. Under an electronic microscope, most rAAV particles appeared full and a few were in intermediate form. Empty particles were rarely seen. The purified rAAV-GFP stocks have been successfully used in in vitro and in vivo transfection experiments. Therefore, this new method offers a simple, rapid and cost-effective way for large-scale rAAV purification.
文摘前期杂交优化后赤芝菌种经液体深层发酵后,提取灵芝菌丝体多糖,并过DEAE-Sepharose Fast Flow柱分离纯化,利用高效体积排阻色谱(HPSEC)检测多糖级分的纯度,采用完全酸水解PMP柱前衍生化RP—HPLC测定多糖级分的单糖组成,多角度光散射仪联用装置(SEC—MALLS)测定其绝对重均分子量(Mw),并且根据分子旋转半径与分子摩尔数的关系曲线斜率初步推断其空间构象。结果显示:分离纯化得到3个多糖级分GLMP1、GLMP2和GLMP3,HPSEC检测其峰面积百分比分别为93.58%,97.64%,99.19%,单糖组成分析结果表明GLMP1、GLMP2和GLMP3均含有甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖和岩藻糖,但单糖摩尔比各异。SEC—MALLS测试GLMP1、GLMP2和GLMP3的Mw分别为4.526×105,4.603×104,3.760×103 g/mol,3个多糖级分构象可能均为高度紧缩且具有分支结构的聚合物。
文摘采用Sepharose CL-6B凝胶柱纯化壶瓶枣多糖(polysaccharides from Zizyphus jujube Mill.cv.Hupingzao,简称ZJP)ZJP-2和ZJP-5组分,并对纯化后多糖的结构进行分析。结果表明:经纯化后得到ZJP-2b和ZJP-5a两种组分均一的壶瓶枣活性多糖,分子质量分别为89.21、61.60 k D,均具备多糖的特征吸收峰,且均以β-构型的吡喃糖为主;ZJP-2b中单糖组成主要为鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖,其物质的量比为32.4∶9.5∶9.4∶14.7∶9.7,而ZJP-5a中单糖组成主要为鼠李糖、阿拉伯糖、木糖、甘露糖和半乳糖,其物质的量比为20.2∶42.9∶2.2∶7.5∶14.5;当质量浓度为3.5 mg/mL时,ZJP-2b和ZJP-5a的羟自由基清除率分别为30.51%和57.22%。
文摘A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion ex- change chromatography on DEAE-Sephadex As0. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60~C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low Ki value of 0.2 ~tM. The inhibitoi- was found to be susceptible to varying concentrations of reducing agents like DTT and 2- mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth ofPieris brassi- cae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.
