穿膜肽-β-葡萄糖苷酶偶联物制备鉴定及激活苦杏仁苷杀伤膀胱癌细胞的初步研究
发布时间：2019-06-07 19:31
【摘要】：目的： 探讨应用异型双功能蛋白交联剂N-唬拍酞胺-3-(2-毗吮二硫)-丙酸醋（SPDP）制备穿膜肽-β-葡萄糖苷酶偶联物的可行性并鉴定其活性、偶联方式。并初步研究穿膜肽-β-葡萄糖苷酶偶联物联合苦杏仁苷体外对人膀胱癌EJ细胞的杀伤作用。为进一步构建高效性穿膜肽-β-葡萄糖苷酶-抗体酶-前药系统提供实验基础。 方法： 1、复苏本课题组前期实验构建并鉴定测序后的高表达穿膜肽-增强型绿色荧光蛋白融合蛋白（CCPs-EGFP）大肠杆菌菌株，对菌株并进行大量培养、诱导表达融合蛋白。采用Ni-NAT Superflow Cartridge层析柱分离纯化；采用还原型聚丙烯酰胺凝胶电泳（SDS-PAGE）对融合蛋白分子量大小以及纯度进行鉴定；利用BCA法蛋白浓度测定试剂盒测定纯化后融合蛋白的浓度；将融合蛋白与膀胱癌EJ细胞共同孵育,置于荧光显微镜下观察，鉴定其穿膜活性。 2、采用异型双功能试剂N一琥珀酰亚胺基一3一(2一吡啶二硫)一丙酸酯（SPDP）交联法将穿膜肽-增强型绿色荧光蛋白融合蛋白（CCPs-EGFP）、β-葡萄糖苷酶进行共价交联，，先利用Ni-NAT Superflow Cartidge层析柱进行第一步分离纯化，再利用Sephadex G-75层析柱进行第二步分离纯化。采用还原型和非还原型聚丙烯酰胺凝胶电泳（SDS-PAGE）对偶联物交联程度、交联方式进行评价及鉴定；将穿膜肽-β-葡萄糖苷酶偶联物与膀胱癌 EJ细胞共同孵育，然后置于荧光显微镜下观察，对偶联物穿膜活性进行鉴定；采用β-葡萄糖苷酶测试盒（DBGD-100）鉴定其酶活性。 3、采用MTT法测定穿膜肽-β-葡萄糖苷酶偶联物联合苦杏仁苷体外对膀胱癌EJ细胞生长的抑制作用。 结果： 1、大量表达、纯化出穿膜肽-增强型绿色荧光蛋白融合蛋白，目的融合蛋白为可溶性、绿色融合蛋白。经还原型聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定目的蛋白分子量大小约为31KD，纯度较高，无明显的杂带。经BCA法蛋白浓度测定试剂盒测定纯化后融合蛋白的浓度为10.723帲痬l。将融合蛋白与膀胱癌EJ细胞共同孵育60分钟后,在荧光显微镜下可观察到膀胱癌EJ细胞内有绿色荧光。 2、穿膜肽-β-葡萄糖苷酶偶联物SDS-PAGE非还原型电泳高分子量端可见明显条带，而还原型电泳分别在穿膜肽和β-葡萄糖苷酶分子量相应位置可见明显条带；穿膜肽-β-葡萄糖苷酶偶联物与膀胱癌EJ细胞共同孵育60分钟后，在荧光显微镜下可见明显绿色荧光；经β-葡萄糖苷酶测试盒（DBGD-100）测定,偶联物仍然保持有良好的酶活性，约为同等浓度β-葡萄糖苷酶活性的80%。 3、经MTT法测定，穿膜肽-β-葡萄糖苷酶偶联物联合苦杏仁苷体外对膀胱癌EJ细胞生长的抑制作用强于单纯β-葡萄糖苷酶联合苦杏仁苷的作用。 结论： 成功大量表达、纯化出穿膜肽-增强型绿色荧光蛋白融合蛋白。利用异型双功能蛋白交联剂SPDP可将该融合蛋白与β-葡萄糖苷酶成功的偶联在一起。分别利用Ni-NAT Superflow Cartidge和Sephadex G-75层析柱可对偶联物进行成功纯化，纯化后穿膜肽-β-葡萄糖苷酶偶联物仍然保持有良好穿膜活性和酶活性。穿膜肽-β-葡萄糖苷酶偶联物联合苦杏仁苷，在体外可提高对膀胱癌EJ细胞的杀伤作用。该方法可能会解决酶-前药系统分子量大药物难以进入细胞内的问题，并为进一步构建高效性穿膜肽-β-葡萄糖苷酶-抗体酶-前药系统提供了实验基础。
[Abstract]:Purpose: To study the feasibility of the preparation of a membrane-penetrating peptide-1-grape-glycosidase conjugate by using a special-shaped double-functional protein cross-linking agent N-triammine-3-(2-bis-dithio)-propionic acid vinegar (SPDP) and to identify the activity, the coupling side, Expression of human bladder cancer EJ cells in vitro and in vitro in ord to provide that experimental basis for the further construction of high-efficiency membrane-penetrating peptide-1-grape-glucoamylase-anti-body-enzyme-prodrug system No, no, no. Methods:1. The high-expression penetrating-membrane peptide-enhanced green fluorescent protein fusion protein (CCPs-EGFP) E. coli strain was constructed and identified in the early stage of the research group. The fusion protein is separated and purified by adopting a Ni-NAT Superflow Carridge chromatography column, the molecular weight and the purity of the fusion protein are identified by adopting the reduced type polyfluoroamine gel electrophoresis (SDS-PAGE), and the purified fusion protein is determined by using the BCA method protein concentration measurement kit. the concentration of the protein; the fusion protein is incubated with the human bladder cancer EJ cell, and the fusion protein is placed under a fluorescence microscope to be observed and identified; And the membrane-penetrating peptide-enhanced green fluorescent protein fusion protein (CCPs-EGFP), HCO3-glucose and the like are prepared by a cross-linking method of a special-shaped double-functional reagent N-succinimino-3-(2-dithio-disulfide)--one propionic acid ester (SPDP) crosslinking method. The enzyme is covalently cross-linked, the first step is first separated and purified by using a Ni-NAT Superflow Cartige column, and then a Sephadex G-75 chromatographic column is used for carrying out the separation and purification. And the second step is to separate and purify. The cross-linking degree and the cross-linking mode of the conjugate are evaluated and identified by using reduced and non-reduced polyfluoroamine gel electrophoresis (SDS-PAGE), and the membrane-penetrating peptide-1-glucose is added. The enzyme conjugate is incubated with the bladder cancer EJ cells and then placed The membrane activity of the conjugate was identified under a fluorescence microscope, and the cassette (DBGD-1) was tested using a yeast-grape glycanase test box (DBGD-1). 00) Identification of its enzyme activity.3. MTT assay was used for the determination of bladder cancer in vitro by the combination of the membrane-penetrating peptide-1-grape glycanase conjugate and the almonds. EJ缁
本文编号：2495029
[Abstract]:Purpose: To study the feasibility of the preparation of a membrane-penetrating peptide-1-grape-glycosidase conjugate by using a special-shaped double-functional protein cross-linking agent N-triammine-3-(2-bis-dithio)-propionic acid vinegar (SPDP) and to identify the activity, the coupling side, Expression of human bladder cancer EJ cells in vitro and in vitro in ord to provide that experimental basis for the further construction of high-efficiency membrane-penetrating peptide-1-grape-glucoamylase-anti-body-enzyme-prodrug system No, no, no. Methods:1. The high-expression penetrating-membrane peptide-enhanced green fluorescent protein fusion protein (CCPs-EGFP) E. coli strain was constructed and identified in the early stage of the research group. The fusion protein is separated and purified by adopting a Ni-NAT Superflow Carridge chromatography column, the molecular weight and the purity of the fusion protein are identified by adopting the reduced type polyfluoroamine gel electrophoresis (SDS-PAGE), and the purified fusion protein is determined by using the BCA method protein concentration measurement kit. the concentration of the protein; the fusion protein is incubated with the human bladder cancer EJ cell, and the fusion protein is placed under a fluorescence microscope to be observed and identified; And the membrane-penetrating peptide-enhanced green fluorescent protein fusion protein (CCPs-EGFP), HCO3-glucose and the like are prepared by a cross-linking method of a special-shaped double-functional reagent N-succinimino-3-(2-dithio-disulfide)--one propionic acid ester (SPDP) crosslinking method. The enzyme is covalently cross-linked, the first step is first separated and purified by using a Ni-NAT Superflow Cartige column, and then a Sephadex G-75 chromatographic column is used for carrying out the separation and purification. And the second step is to separate and purify. The cross-linking degree and the cross-linking mode of the conjugate are evaluated and identified by using reduced and non-reduced polyfluoroamine gel electrophoresis (SDS-PAGE), and the membrane-penetrating peptide-1-glucose is added. The enzyme conjugate is incubated with the bladder cancer EJ cells and then placed The membrane activity of the conjugate was identified under a fluorescence microscope, and the cassette (DBGD-1) was tested using a yeast-grape glycanase test box (DBGD-1). 00) Identification of its enzyme activity.3. MTT assay was used for the determination of bladder cancer in vitro by the combination of the membrane-penetrating peptide-1-grape glycanase conjugate and the almonds. EJ缁
本文编号：2495029
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