穿膜肽-β-葡萄糖苷酶—西妥昔单抗偶联物的制备与鉴定

发布时间：2018-05-21 06:03

本文选题：穿膜肽 + 量子点　；参考：《广西医科大学》2015年硕士论文

【摘要】：目的探讨应用异型双功能蛋白偶联剂衍生物硫代琥珀酰亚胺基4-(N-马来酰亚胺甲基)-环己烷-1-羧化物(Sulfo-SMCC)制备穿膜肽-p-葡萄糖苷酶-西妥昔单抗偶联物的可行性,同时检测偶联物中的穿膜肽活性、p-葡萄糖苷酶活性及西妥昔单抗活性,为下一步研究其抗肿瘤效应奠定了基础。方法1、采用Sulfo-SMCC将氨基水溶性量子点与巯基化穿膜肽进行共价交联。将偶联后的穿膜肽-量子点和人膀胱癌EJ细胞共培养,检测穿膜肽的活性。2、应用硫代琥珀酰亚胺基4-(N-马来酰亚胺甲基)-环己烷-1-羧化物(Sulfo-SMCC)将西妥昔单抗、p-葡萄糖苷酶进行偶联。采用SDS-PAGE、比色法、NHS-Alexa Fluor 488标记偶联物进行鉴定二联偶联物并检测β-葡萄糖苷酶、西妥昔单抗的活性。3、采用同上方法将穿膜肽、p-葡萄糖苷酶、西妥昔单抗进行共价偶联,制备穿膜肽-β-葡萄糖苷酶-西妥昔单抗三联偶联物。采用非还原型聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定三联偶联物的连接方式和成份；比色法鉴定三联偶联物酶活性；采用NHS-Alexa Fluor 488对偶联物进行荧光标记,然后将穿膜肽-β-葡萄糖苷酶-西妥昔单抗三联物和EJ细胞共培养,检测三联偶联物中的穿膜肽活性及抗体活性。结果1、穿膜肽活性鉴定结果：将穿膜肽-量子点与EJ细胞分别共培养1小时和2小时后,在Olympus BX51正置荧光显微镜下观察到随着时间的延长,人膀胱癌EJ细胞内的红色荧光越来越明显,而对照组未见荧光,说明穿膜肽具有良好的穿膜活性。2、西妥昔单抗-β-葡萄糖苷酶偶联物的鉴定结果：SDS-PAGE结果示在3泳道高分子端可见清晰的条带,而在β-葡萄糖苷酶、西妥昔单抗相对应位置未见条带。比色法结果示,等浓度二联物的酶活性低于β-葡萄糖苷酶的酶活性(U/L：669.56±39.23 vs 870.60±61.34,t=7.863,P0.05),二联物仍然具有良好酶活性。标记有绿色荧光的西妥昔单抗-β-葡萄糖苷酶二联物与EJ细胞共培养1小时后,置于Olympus BX51正置荧光显微镜下观察到细胞周围有绿色荧光,说明偶联物仍然保持良好的抗体活性；3、穿膜肽-β-葡萄糖苷酶-西妥昔单抗偶联物的鉴定结果：在电泳图上可见到4泳道高分子端的清晰条带,而在β-葡萄糖苷酶、穿膜肽-β-葡萄糖苷酶、西妥昔单抗相对应位置未见条带出现。比色法测定结果显示,等浓度三联物的酶活性低于β-葡萄糖苷酶(U/L:612.75110.58 vs 917.03±22.72,t=21.02,P0.05),但三联物仍然保持有一定的酶活性。标记有绿色荧光染料的穿膜肽-β-葡萄糖苷酶-西妥昔单抗三联物与EJ细胞共培养1小时后,在Olympus BX51正置荧光显微镜下观察到EJ细胞内与周围有明显的绿色荧光,说明偶联物仍保持良好的穿膜活性与抗体活性；结论Sulfo-SMCC可以将穿膜肽、β-葡萄糖苷酶、西妥昔单抗成功的偶联在一起,同时三联物仍能保持良好的穿膜肽活性、酶活性与抗体活性。该体系的建立为进一步研究其抗肿瘤效应奠定了基础,为抗体介导的酶前体药物疗法(ADEPT)的开发提供了新的思路,并且对浸润性肿瘤的治疗具有重要意义。
[Abstract]:Objective To investigate the feasibility of using heterobifunctional protein coupling agent derivative thiosuccinimide - 4 - ( N - maleimide methyl ) - cyclohexane - 1 - carboxylate ( Sulfo - SMCC ) to prepare the membrane - penetrating peptide - p - glucosidase - cetuximab conjugate .
identification of enzyme activity of triplet conjugate by colorimetric method ;
Results 1 . The results showed that the red fluorescence in human bladder cancer cell line was more and more obvious with the time extension . The results showed that the activity of 尾 - glucosidase was lower than that of 尾 - glucosidase ( U / L : 669.56 卤 39.23 vs 870.60 卤 61.34 , t = 7.863 , P0.05 ) .
3 . The results showed that the clear bands of 4 - lane polymer could be seen on the electrophoresis map , while the corresponding sites of 尾 - glucosidase , transmembrane peptide - 尾 - glucosidase and cetuximab were not observed . The results showed that the enzyme activity of the three - linked immunosorbent assay was lower than that of 尾 - glucosidase ( U / L : 612 . 75110.58 vs 917.03 卤 22.72 , t = 21.02 , P0.05 ) .
Conclusion Sulfo - SMCC can successfully couple with membrane - penetrating peptide , 尾 - glucosidase and cetuximab , and it can maintain good membrane - penetrating peptide activity , enzyme activity and antibody activity . The establishment of the system lays a foundation for further research on its anti - tumor effect , provides a new idea for the development of antibody - mediated enzyme prodrug therapy ( ADEPT ) , and has important significance for the treatment of invasive tumors .
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943;R927

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