葛根素特异性敲除技术方法及应用研究
摘要
目的：制备葛根素单克隆抗体,并研究其应用范围及方法。
1合成葛根素-牛血清白蛋白(免疫原)与葛根素-多聚赖氨酸(包被原)偶联物,并制备葛根素单克隆抗体。
2利用葛根素单克隆抗体建立葛根素酶联免疫吸附法,考察准确性、重复性、重现性及加样回收率,并进行药品及生物样品中葛根素含量的测定。
3制备葛根素免疫亲和层析柱,优化敲除、洗脱工艺参数,考察凝胶抗体偶联比、柱容量、回收率,建立敲除样品指纹图谱。
4利用免疫亲和层析柱敲除葛根素,运用对比研究方法进行葛根素在葛根抗炎效果中的作用评价
5进行葛根素单克隆抗体荧光标记,利用人脐静脉内皮细胞初步探索葛根素在细胞上的作用靶点。
方法：脾细胞融合制备葛根素单克隆抗体,免疫亲和层析柱进行葛根素敲除并研究葛根素药效,荧光标记法进行葛根素在细胞上的作用靶点发现。
1高碘酸钠氧化法合成葛根素与载体蛋白的偶联物作为包被原(葛根素-多聚赖氨酸)及免疫原(葛根素-牛血清白蛋白),免疫后进行血清抗体检测,经过脾细胞融合,阳性杂交瘤细胞筛选,单克隆化后,腹水诱导法大量制备腹水,辛酸硫酸铵法及蛋白G柱法纯化葛根素单克隆抗体,并计算交叉反应率进行抗体特异性检测。
2优化包被原包被浓度、一抗工作浓度、线性检测范围常规间接竞争ELISA法建立葛根素相关的酶联免疫吸附法。并考察其准确性、重复性、重现性及加样回收率,进行药品及生物样品中葛根素含量的测定,与常规HPLC法检测结果比较,流动相为甲醇40%：水60%,并进行相关性分析。
3利用葛根素单克隆抗体,经琼脂糖凝胶活化、抗体偶联、封闭、洗杂、平衡后,装柱并制备葛根素免疫亲和层析柱。优化筛选上样缓冲液、敲除缓冲液和洗脱缓冲液以及流速条件,通过考察抗体凝胶偶联比、柱容量、回收率评价已制备的葛根素免疫亲和层析柱。建立敲除样品的指纹图谱,条件为：安捷伦1260,安捷伦ZORBAX SBC-18柱,进样量10-μL,流速1mL/min,柱温25℃流动相为甲醇水溶液,梯度浓度为0-10min甲醇由0-23%,10-30min甲醇浓度保持23%,30-50min甲醇由23-35%,50-60min甲醇由35-70%。
4通过葛根素免疫亲和层析柱敲除葛根提取液中的葛根素,以LPS诱导巨噬细胞分泌IL-1β及NO,通过葛根素敲除前后药效对比研究,观察葛根素在葛根对白介素1β及一氧化氮的抑制效果中的作用。
5利用荧光蛋白标记法进行葛根素单克隆抗体的荧光标记,人脐静脉内皮细胞经10%小鼠血清封闭1小时后,葛根素1μ g/mL培养1小时,加入荧光标记抗体(1：100稀释)孵育半小时后经荧光倒置显微镜观察荧光标记情况。
结果：经过性能检测,葛根素单克隆抗体制备成功,所建立的葛根素酶联免疫吸附法及免疫亲和层析柱性能稳定,精密度高,回收率好,重复性均符合要求,制备成功,可用于葛根素的药理药效研究,荧光标记成功,初步判断葛根素在人脐静脉内皮细胞中的作用靶点位于细胞核膜上。
1葛根素人工抗原合成成功,经紫外检测并计算得出其偶联比为6：1,经融合筛选后成功制备出分泌抗葛根素单克隆抗体的细胞株AA9,所分泌抗体特异性强,除与黄芩素有51.8%的交叉反应外,与其余结构类似物均无明显交叉反应(交叉反应率均<0.01)。
2利用葛根素单克隆抗体成功建立了酶联免疫吸附法,包被原浓度为1μ g/mL,抗体工作浓度为10ng/mL,线性竞争范围为10ng-1.28μ g,灵敏度(50%抑制时的浓度)为128ng/mL,线性方程为：y=-0.156ln(x)+1.2804,R2=0.997。准确性及重复性良好,孔间差变异系数<3%,板间差变异系数<7.5%,平均加样回收率为101.4%,可以用于样品检测。
3制备了葛根素免疫亲和层析柱,并对上样敲除洗脱条件进行了优化,上样及敲除缓冲液均选为蒸馏水,洗脱缓冲液选为30%甲醇,抗体偶联率为98%,抗体凝胶偶联比为13.5mg/mL,柱容量为13.6μ g/mL.并制备了样品及敲除提取液的HPLC指纹图谱,成功建立了葛根素敲除法。
4利用敲除法评价葛根素在葛根抗炎效果中的作用。将处于对数生长期的巨噬细胞分为空白对照组(C)、模型对照组(M)、给药组：葛根提取液组(PLRE)、葛根素敲除组(PU-KO-PLRE)和葛根素组(PU)。培养的巨噬细胞经LPS诱导后NO及IL-1β分泌明显增加,而葛根素明显抑制IL-1β分泌,敲除后则明显不能抑制,说明葛根对LPS诱导的IL-1β分泌抑制作用主要为葛根素产生。
5葛根素单克隆抗体经过蛋白G纯化后,用荧光蛋白进行标记。人脐静脉内皮细胞培养至对数生长期后分为空白对照组(C)、葛根素对照组(PU)、抗体对照组(MAb)、葛根素加标记抗体组(P+M),按组别分别给予标记抗体或葛根素后充分孵育,荧光倒置显微镜观察到,空白对照组、葛根素对照组、标记抗体组均无特征性荧光产生,而葛根素加标记抗体组可观察到细胞核膜上有明显荧光产生。
讨论：高碘酸钠法合成葛根素效果良好,葛根素单抗灵敏度高,能用于葛根素的微量分析,比常规的HPLC方法更加快速准确,前处理方法更加简化。免疫亲和层析柱可用于中药提取液或复方中的物质敲除,借助敲除前后的比较研究可证明活性物质在药材或复方中的真正药理作用与贡献度。
Objective:Preparation of puerarin monoclonal antibodies, and its application study.
1Synthesize puerarin-bovine serum albumin (immunogen) and puerarin poly-lysine (including the original) conjugate and monoclonal antibody preparation puerarin.
2Use of puerarin monoclonal antibody to puerarin enzyme-linked immunosorbent assay, inspection accuracy, repeatability, reproducibility and recoveries, and the Determination of puerarin in pharmaceutical and biological samples.
3Preparation of puerarin immunoaffinity chromatography column, optimizing the knockout, the elution process parameters, examine gel antibody coupling ratio, column capacity, recovery, the knockout samples fingerprinting.
4immunoaffinity chromatography column knockout puerarin, puerarin the role of evaluation in the anti-inflammatory effects of Pueraria use of contrast research methods.
5puerarin monoclonal antibody fluorescently labeled puerarin on the cell target of preliminary exploration, the use of human umbilical vein endothelial cells.
Methods:Preparation of puerarin monoclonal antibody immunoaffinity chromatography and puerarin knock addition to puerarin efficacy, fluorescence labeling method puerarin on the cell target discovery.
1Sodium periodate oxidation synthesis puerarin carrier protein conjugates as a package by the original (puerarin-poly-L-lysine) and immunogen (puerarin-bovine serum albumin), immune serum antibodies, after splenic cell fusion, the positive hybridoma screening, monoclonal after ascites was induced large scale preparation of ascites, bitterness ammonium sulfate precipitation and protein G column purified puerarin monoclonal antibody, and calculate the cross-reactivity to antibodies specific detection.
2optimization package is the original concentration, the concentration of an anti-linear detection range of conventional indirect competitive ELISA method puerarin enzyme-linked immunosorbent assay. And examine its accuracy, repeatability, reproducibility and recoveries, pharmaceutical and biological samples Determination of puerarin, compared with conventional HPLC assay results, the mobile phase of40%methanol:60%water, and correlation analysis.
3puerarin monoclonal antibody by agarose gel activation, antibody-coupled, closed, wash Miscellaneous balance, column packing and preparation the puerarin immune affinity chromatography. Optimization of the screening sample buffer, knockout buffer and elution buffer and flow rate conditions, by examining the antibody gel coupling ratio, the capacity of the column, the recovery rate evaluation prepared puerarin immunoaffinity chromatography column. Establish the knockout sample fingerprints conditions:Agilent1260the Agilent ZORBAX SBC-18column, the injection volume was10ul flow rate of1mL/min, column temperature25℃, mobile phase was methanol aqueous solution, the gradient of concentration of0-10min. methanol methanol concentration by0-23%,10-30min to maintain23%,30-50min methanol methanol from23-35%,50-60min by35-70%.
4puerarin immunoaffinity chromatography knockout kudzu root extract puerarin solution to LPS-induced macrophages to secrete IL-lbeta and NO efficacy by puerarin knockout before and after contrast research, observation of puerarin in Pueraria dialogue interleukin-lbeta and nitric oxide's role in the inhibitory effect.
5fluorescent protein puerarin fluorescently labeled monoclonal antibodies, human umbilical vein endothelial cells blocked for1hour in10%serum, incubated for1hour puerarin1μg/mL fluorescently labeled antibody (1:100dilution) and incubated for half an hour after an inverted fluorescence microscope to observe the fluorescent labeling.
Results:After performance testing, puerarin monoclonal antibodies were successfully established by the puerarin enzyme-linked immunosorbent assay and immunoaffinity chromatography and stable performance, high precision, good recovery, repeatability to meet the requirements, preparation, can be usedat the puerarin the pharmacological efficacy studies, fluorescently labeled successful, determine the initial targets of puerarin in human umbilical vein endothelial cells located in the nuclear membrane.
1puerarin artificial antigen successfully synthesized by UV detector and calculated the coupling ratio of6:1, were successfully prepared by fusion screening cell lines secrete anti-puerarin monoclonal antibody AA9secreted antibody specificity, In addition to51.8%cross-reactivity with skullcap known, no significant cross-reactivity (cross-reactivity ratios<0.01), with the remaining structural analogues.
2puerarin monoclonal antibody successfully established enzyme-linked immunosorbent assay, the original concentration to1microg/mL, the antibody concentration of10ng/mL, linear competitive range of10ng-1.28μg, sensitivity (50%inhibition concentration) of128ng/mL, the linear equation:y=-0.156In (x)+1.2804, R2=0.997. Good accuracy and repeatability, hole difference between the coefficient of variation<3%, the the plate difference between the coefficient of variation<7.5%, the average recovery was101.4%, can be used for sample testing.
3Preparation of the puerarin immunoaffinity chromatography, knocking elution conditions were optimized and the sample on the sample and the buffer are knockout preferably distilled water,30%methanol elution buffer preferably, the antibody-coupled was98%, the antibody gel Coupling13.5mg/mL, column capacity13.6mg/mL. And preparation of samples and knock In addition to extract HPLC fingerprint successfully established the puerarin knock division.
4knock division evaluation of the role of puerarin in Pueraria anti-inflammatory effect. Will be in the logarithmic growth phase macrophages divided into control group (C), model group (M), administered group:Pueraria lobata solution group (plre), puerarin knockout group (PU-KO-plre) and the the puerarin group of (PU). Cultured macrophages by LPS-induced NO and IL-1beta secretion increased significantly, puerarin significantly inhibited IL-1beta secretion can not be suppressed significantly in the knockout Pueraria on LPS-induced IL-1beta secretion inhibition The puerarin produce.
5puerarin after purified by protein G monoclonal antibody to be labeled with a fluorescent protein. Human umbilical vein endothelial cells cultured to logarithmic phase divided into control group (C), the puerarin control group (PU), antibody control group (MAb) labeled antibody puerarin group (P+M), by group do not give a labeled antibody or puerarin fully incubated inverted fluorescence microscope to the control group, puerarin control group labeled antibody group had no characteristic fluorescence puerarin labeled antibody group can be observed to the nuclear membrane There are significant fluorescence.
Conclusion:Effect of the sodium periodate synthesis puerarin, puerarin monoclonal antibody with high sensitivity, puerarin can be used for trace analysis more quickly and accurately than conventional HPLC methods, the former approach is more streamlined. Immune affinity chromatography can be used in traditional Chinese medicine extraction or the compound material knockout, with the knock on active substances in herbs or compound addition to the before and after comparison study to prove the true pharmacological effects and the contribution.
1合成葛根素-牛血清白蛋白(免疫原)与葛根素-多聚赖氨酸(包被原)偶联物,并制备葛根素单克隆抗体。
2利用葛根素单克隆抗体建立葛根素酶联免疫吸附法,考察准确性、重复性、重现性及加样回收率,并进行药品及生物样品中葛根素含量的测定。
3制备葛根素免疫亲和层析柱,优化敲除、洗脱工艺参数,考察凝胶抗体偶联比、柱容量、回收率,建立敲除样品指纹图谱。
4利用免疫亲和层析柱敲除葛根素,运用对比研究方法进行葛根素在葛根抗炎效果中的作用评价
5进行葛根素单克隆抗体荧光标记,利用人脐静脉内皮细胞初步探索葛根素在细胞上的作用靶点。
方法：脾细胞融合制备葛根素单克隆抗体,免疫亲和层析柱进行葛根素敲除并研究葛根素药效,荧光标记法进行葛根素在细胞上的作用靶点发现。
1高碘酸钠氧化法合成葛根素与载体蛋白的偶联物作为包被原(葛根素-多聚赖氨酸)及免疫原(葛根素-牛血清白蛋白),免疫后进行血清抗体检测,经过脾细胞融合,阳性杂交瘤细胞筛选,单克隆化后,腹水诱导法大量制备腹水,辛酸硫酸铵法及蛋白G柱法纯化葛根素单克隆抗体,并计算交叉反应率进行抗体特异性检测。
2优化包被原包被浓度、一抗工作浓度、线性检测范围常规间接竞争ELISA法建立葛根素相关的酶联免疫吸附法。并考察其准确性、重复性、重现性及加样回收率,进行药品及生物样品中葛根素含量的测定,与常规HPLC法检测结果比较,流动相为甲醇40%：水60%,并进行相关性分析。
3利用葛根素单克隆抗体,经琼脂糖凝胶活化、抗体偶联、封闭、洗杂、平衡后,装柱并制备葛根素免疫亲和层析柱。优化筛选上样缓冲液、敲除缓冲液和洗脱缓冲液以及流速条件,通过考察抗体凝胶偶联比、柱容量、回收率评价已制备的葛根素免疫亲和层析柱。建立敲除样品的指纹图谱,条件为：安捷伦1260,安捷伦ZORBAX SBC-18柱,进样量10-μL,流速1mL/min,柱温25℃流动相为甲醇水溶液,梯度浓度为0-10min甲醇由0-23%,10-30min甲醇浓度保持23%,30-50min甲醇由23-35%,50-60min甲醇由35-70%。
4通过葛根素免疫亲和层析柱敲除葛根提取液中的葛根素,以LPS诱导巨噬细胞分泌IL-1β及NO,通过葛根素敲除前后药效对比研究,观察葛根素在葛根对白介素1β及一氧化氮的抑制效果中的作用。
5利用荧光蛋白标记法进行葛根素单克隆抗体的荧光标记,人脐静脉内皮细胞经10%小鼠血清封闭1小时后,葛根素1μ g/mL培养1小时,加入荧光标记抗体(1：100稀释)孵育半小时后经荧光倒置显微镜观察荧光标记情况。
结果：经过性能检测,葛根素单克隆抗体制备成功,所建立的葛根素酶联免疫吸附法及免疫亲和层析柱性能稳定,精密度高,回收率好,重复性均符合要求,制备成功,可用于葛根素的药理药效研究,荧光标记成功,初步判断葛根素在人脐静脉内皮细胞中的作用靶点位于细胞核膜上。
1葛根素人工抗原合成成功,经紫外检测并计算得出其偶联比为6：1,经融合筛选后成功制备出分泌抗葛根素单克隆抗体的细胞株AA9,所分泌抗体特异性强,除与黄芩素有51.8%的交叉反应外,与其余结构类似物均无明显交叉反应(交叉反应率均<0.01)。
2利用葛根素单克隆抗体成功建立了酶联免疫吸附法,包被原浓度为1μ g/mL,抗体工作浓度为10ng/mL,线性竞争范围为10ng-1.28μ g,灵敏度(50%抑制时的浓度)为128ng/mL,线性方程为：y=-0.156ln(x)+1.2804,R2=0.997。准确性及重复性良好,孔间差变异系数<3%,板间差变异系数<7.5%,平均加样回收率为101.4%,可以用于样品检测。
3制备了葛根素免疫亲和层析柱,并对上样敲除洗脱条件进行了优化,上样及敲除缓冲液均选为蒸馏水,洗脱缓冲液选为30%甲醇,抗体偶联率为98%,抗体凝胶偶联比为13.5mg/mL,柱容量为13.6μ g/mL.并制备了样品及敲除提取液的HPLC指纹图谱,成功建立了葛根素敲除法。
4利用敲除法评价葛根素在葛根抗炎效果中的作用。将处于对数生长期的巨噬细胞分为空白对照组(C)、模型对照组(M)、给药组：葛根提取液组(PLRE)、葛根素敲除组(PU-KO-PLRE)和葛根素组(PU)。培养的巨噬细胞经LPS诱导后NO及IL-1β分泌明显增加,而葛根素明显抑制IL-1β分泌,敲除后则明显不能抑制,说明葛根对LPS诱导的IL-1β分泌抑制作用主要为葛根素产生。
5葛根素单克隆抗体经过蛋白G纯化后,用荧光蛋白进行标记。人脐静脉内皮细胞培养至对数生长期后分为空白对照组(C)、葛根素对照组(PU)、抗体对照组(MAb)、葛根素加标记抗体组(P+M),按组别分别给予标记抗体或葛根素后充分孵育,荧光倒置显微镜观察到,空白对照组、葛根素对照组、标记抗体组均无特征性荧光产生,而葛根素加标记抗体组可观察到细胞核膜上有明显荧光产生。
讨论：高碘酸钠法合成葛根素效果良好,葛根素单抗灵敏度高,能用于葛根素的微量分析,比常规的HPLC方法更加快速准确,前处理方法更加简化。免疫亲和层析柱可用于中药提取液或复方中的物质敲除,借助敲除前后的比较研究可证明活性物质在药材或复方中的真正药理作用与贡献度。
Objective:Preparation of puerarin monoclonal antibodies, and its application study.
1Synthesize puerarin-bovine serum albumin (immunogen) and puerarin poly-lysine (including the original) conjugate and monoclonal antibody preparation puerarin.
2Use of puerarin monoclonal antibody to puerarin enzyme-linked immunosorbent assay, inspection accuracy, repeatability, reproducibility and recoveries, and the Determination of puerarin in pharmaceutical and biological samples.
3Preparation of puerarin immunoaffinity chromatography column, optimizing the knockout, the elution process parameters, examine gel antibody coupling ratio, column capacity, recovery, the knockout samples fingerprinting.
4immunoaffinity chromatography column knockout puerarin, puerarin the role of evaluation in the anti-inflammatory effects of Pueraria use of contrast research methods.
5puerarin monoclonal antibody fluorescently labeled puerarin on the cell target of preliminary exploration, the use of human umbilical vein endothelial cells.
Methods:Preparation of puerarin monoclonal antibody immunoaffinity chromatography and puerarin knock addition to puerarin efficacy, fluorescence labeling method puerarin on the cell target discovery.
1Sodium periodate oxidation synthesis puerarin carrier protein conjugates as a package by the original (puerarin-poly-L-lysine) and immunogen (puerarin-bovine serum albumin), immune serum antibodies, after splenic cell fusion, the positive hybridoma screening, monoclonal after ascites was induced large scale preparation of ascites, bitterness ammonium sulfate precipitation and protein G column purified puerarin monoclonal antibody, and calculate the cross-reactivity to antibodies specific detection.
2optimization package is the original concentration, the concentration of an anti-linear detection range of conventional indirect competitive ELISA method puerarin enzyme-linked immunosorbent assay. And examine its accuracy, repeatability, reproducibility and recoveries, pharmaceutical and biological samples Determination of puerarin, compared with conventional HPLC assay results, the mobile phase of40%methanol:60%water, and correlation analysis.
3puerarin monoclonal antibody by agarose gel activation, antibody-coupled, closed, wash Miscellaneous balance, column packing and preparation the puerarin immune affinity chromatography. Optimization of the screening sample buffer, knockout buffer and elution buffer and flow rate conditions, by examining the antibody gel coupling ratio, the capacity of the column, the recovery rate evaluation prepared puerarin immunoaffinity chromatography column. Establish the knockout sample fingerprints conditions:Agilent1260the Agilent ZORBAX SBC-18column, the injection volume was10ul flow rate of1mL/min, column temperature25℃, mobile phase was methanol aqueous solution, the gradient of concentration of0-10min. methanol methanol concentration by0-23%,10-30min to maintain23%,30-50min methanol methanol from23-35%,50-60min by35-70%.
4puerarin immunoaffinity chromatography knockout kudzu root extract puerarin solution to LPS-induced macrophages to secrete IL-lbeta and NO efficacy by puerarin knockout before and after contrast research, observation of puerarin in Pueraria dialogue interleukin-lbeta and nitric oxide's role in the inhibitory effect.
5fluorescent protein puerarin fluorescently labeled monoclonal antibodies, human umbilical vein endothelial cells blocked for1hour in10%serum, incubated for1hour puerarin1μg/mL fluorescently labeled antibody (1:100dilution) and incubated for half an hour after an inverted fluorescence microscope to observe the fluorescent labeling.
Results:After performance testing, puerarin monoclonal antibodies were successfully established by the puerarin enzyme-linked immunosorbent assay and immunoaffinity chromatography and stable performance, high precision, good recovery, repeatability to meet the requirements, preparation, can be usedat the puerarin the pharmacological efficacy studies, fluorescently labeled successful, determine the initial targets of puerarin in human umbilical vein endothelial cells located in the nuclear membrane.
1puerarin artificial antigen successfully synthesized by UV detector and calculated the coupling ratio of6:1, were successfully prepared by fusion screening cell lines secrete anti-puerarin monoclonal antibody AA9secreted antibody specificity, In addition to51.8%cross-reactivity with skullcap known, no significant cross-reactivity (cross-reactivity ratios<0.01), with the remaining structural analogues.
2puerarin monoclonal antibody successfully established enzyme-linked immunosorbent assay, the original concentration to1microg/mL, the antibody concentration of10ng/mL, linear competitive range of10ng-1.28μg, sensitivity (50%inhibition concentration) of128ng/mL, the linear equation:y=-0.156In (x)+1.2804, R2=0.997. Good accuracy and repeatability, hole difference between the coefficient of variation<3%, the the plate difference between the coefficient of variation<7.5%, the average recovery was101.4%, can be used for sample testing.
3Preparation of the puerarin immunoaffinity chromatography, knocking elution conditions were optimized and the sample on the sample and the buffer are knockout preferably distilled water,30%methanol elution buffer preferably, the antibody-coupled was98%, the antibody gel Coupling13.5mg/mL, column capacity13.6mg/mL. And preparation of samples and knock In addition to extract HPLC fingerprint successfully established the puerarin knock division.
4knock division evaluation of the role of puerarin in Pueraria anti-inflammatory effect. Will be in the logarithmic growth phase macrophages divided into control group (C), model group (M), administered group:Pueraria lobata solution group (plre), puerarin knockout group (PU-KO-plre) and the the puerarin group of (PU). Cultured macrophages by LPS-induced NO and IL-1beta secretion increased significantly, puerarin significantly inhibited IL-1beta secretion can not be suppressed significantly in the knockout Pueraria on LPS-induced IL-1beta secretion inhibition The puerarin produce.
5puerarin after purified by protein G monoclonal antibody to be labeled with a fluorescent protein. Human umbilical vein endothelial cells cultured to logarithmic phase divided into control group (C), the puerarin control group (PU), antibody control group (MAb) labeled antibody puerarin group (P+M), by group do not give a labeled antibody or puerarin fully incubated inverted fluorescence microscope to the control group, puerarin control group labeled antibody group had no characteristic fluorescence puerarin labeled antibody group can be observed to the nuclear membrane There are significant fluorescence.
Conclusion:Effect of the sodium periodate synthesis puerarin, puerarin monoclonal antibody with high sensitivity, puerarin can be used for trace analysis more quickly and accurately than conventional HPLC methods, the former approach is more streamlined. Immune affinity chromatography can be used in traditional Chinese medicine extraction or the compound material knockout, with the knock on active substances in herbs or compound addition to the before and after comparison study to prove the true pharmacological effects and the contribution.
引文
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