穿膜肽-β-葡萄糖苷酶偶聯(lián)物制備鑒定及激活苦杏仁苷殺傷膀胱癌細胞的初步研究
發(fā)布時間：2019-06-07 19:31
【摘要】：目的： 探討應(yīng)用異型雙功能蛋白交聯(lián)劑N-唬拍酞胺-3-(2-毗吮二硫)-丙酸醋（SPDP）制備穿膜肽-β-葡萄糖苷酶偶聯(lián)物的可行性并鑒定其活性、偶聯(lián)方式。并初步研究穿膜肽-β-葡萄糖苷酶偶聯(lián)物聯(lián)合苦杏仁苷體外對人膀胱癌EJ細胞的殺傷作用。為進一步構(gòu)建高效性穿膜肽-β-葡萄糖苷酶-抗體酶-前藥系統(tǒng)提供實驗基礎(chǔ)。 方法： 1、復(fù)蘇本課題組前期實驗構(gòu)建并鑒定測序后的高表達穿膜肽-增強型綠色熒光蛋白融合蛋白（CCPs-EGFP）大腸桿菌菌株，對菌株并進行大量培養(yǎng)、誘導(dǎo)表達融合蛋白。采用Ni-NAT Superflow Cartridge層析柱分離純化；采用還原型聚丙烯酰胺凝膠電泳（SDS-PAGE）對融合蛋白分子量大小以及純度進行鑒定；利用BCA法蛋白濃度測定試劑盒測定純化后融合蛋白的濃度；將融合蛋白與膀胱癌EJ細胞共同孵育,置于熒光顯微鏡下觀察，鑒定其穿膜活性。 2、采用異型雙功能試劑N一琥珀酰亞胺基一3一(2一吡啶二硫)一丙酸酯（SPDP）交聯(lián)法將穿膜肽-增強型綠色熒光蛋白融合蛋白（CCPs-EGFP）、β-葡萄糖苷酶進行共價交聯(lián)，，先利用Ni-NAT Superflow Cartidge層析柱進行第一步分離純化，再利用Sephadex G-75層析柱進行第二步分離純化。采用還原型和非還原型聚丙烯酰胺凝膠電泳（SDS-PAGE）對偶聯(lián)物交聯(lián)程度、交聯(lián)方式進行評價及鑒定；將穿膜肽-β-葡萄糖苷酶偶聯(lián)物與膀胱癌 EJ細胞共同孵育，然后置于熒光顯微鏡下觀察，對偶聯(lián)物穿膜活性進行鑒定；采用β-葡萄糖苷酶測試盒（DBGD-100）鑒定其酶活性。 3、采用MTT法測定穿膜肽-β-葡萄糖苷酶偶聯(lián)物聯(lián)合苦杏仁苷體外對膀胱癌EJ細胞生長的抑制作用。 結(jié)果： 1、大量表達、純化出穿膜肽-增強型綠色熒光蛋白融合蛋白，目的融合蛋白為可溶性、綠色融合蛋白。經(jīng)還原型聚丙烯酰胺凝膠電泳（SDS-PAGE）鑒定目的蛋白分子量大小約為31KD，純度較高，無明顯的雜帶。經(jīng)BCA法蛋白濃度測定試劑盒測定純化后融合蛋白的濃度為10.723帲痬l。將融合蛋白與膀胱癌EJ細胞共同孵育60分鐘后,在熒光顯微鏡下可觀察到膀胱癌EJ細胞內(nèi)有綠色熒光。 2、穿膜肽-β-葡萄糖苷酶偶聯(lián)物SDS-PAGE非還原型電泳高分子量端可見明顯條帶，而還原型電泳分別在穿膜肽和β-葡萄糖苷酶分子量相應(yīng)位置可見明顯條帶；穿膜肽-β-葡萄糖苷酶偶聯(lián)物與膀胱癌EJ細胞共同孵育60分鐘后，在熒光顯微鏡下可見明顯綠色熒光；經(jīng)β-葡萄糖苷酶測試盒（DBGD-100）測定,偶聯(lián)物仍然保持有良好的酶活性，約為同等濃度β-葡萄糖苷酶活性的80%。 3、經(jīng)MTT法測定，穿膜肽-β-葡萄糖苷酶偶聯(lián)物聯(lián)合苦杏仁苷體外對膀胱癌EJ細胞生長的抑制作用強于單純β-葡萄糖苷酶聯(lián)合苦杏仁苷的作用。 結(jié)論： 成功大量表達、純化出穿膜肽-增強型綠色熒光蛋白融合蛋白。利用異型雙功能蛋白交聯(lián)劑SPDP可將該融合蛋白與β-葡萄糖苷酶成功的偶聯(lián)在一起。分別利用Ni-NAT Superflow Cartidge和Sephadex G-75層析柱可對偶聯(lián)物進行成功純化，純化后穿膜肽-β-葡萄糖苷酶偶聯(lián)物仍然保持有良好穿膜活性和酶活性。穿膜肽-β-葡萄糖苷酶偶聯(lián)物聯(lián)合苦杏仁苷，在體外可提高對膀胱癌EJ細胞的殺傷作用。該方法可能會解決酶-前藥系統(tǒng)分子量大藥物難以進入細胞內(nèi)的問題，并為進一步構(gòu)建高效性穿膜肽-β-葡萄糖苷酶-抗體酶-前藥系統(tǒng)提供了實驗基礎(chǔ)。
[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緇
本文編號：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緇
本文編號：2495029
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