天津医药

天津医药 ›› 2014, Vol. 42 ›› Issue (12): 1153-1155.doi: 10.3969/j.issn.0253-9896.2014.12.001

• 细胞与分子生物学 •    下一篇

西妥昔单抗-β葡萄糖苷酶偶联物的制备及鉴定

刘云龙1,周洁2,王光文2,聂振2   

  1. 1. 广西医科大学第一附属医院泌尿外科
    2. 广西医科大学第一附属医院
  • 收稿日期:2014-07-03 修回日期:2014-08-20 出版日期:2014-12-15 发布日期:2014-12-15
  • 通讯作者: 周洁 E-mail:zhoujieuser@163.com
  • 基金资助:
    穿膜肽-抗体导向酶解前药系统对膀胱癌作用机制的研究

Preparation and Identification of Cetuximab-β-Glucosidase Conjugates

Liu yunlong, Zhoujie, Wangguangwen, Niezhen   

  • Received:2014-07-03 Revised:2014-08-20 Published:2014-12-15 Online:2014-12-15

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摘要：

【摘要】 目的 制备西妥昔单抗-β葡萄糖苷酶偶联物并鉴定其酶活性与抗体活性。 方法 采用 4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Sulfo-SMCC)将西妥昔单抗、β-葡萄糖苷酶进行共价交联。 采用非还原型聚丙烯酰胺凝胶电泳（SDS-PAGE）、比色法、间接免疫荧光法对偶联物进行鉴定并检测β-葡萄糖苷酶、西妥昔单抗的活性。 结果 在电泳图上可分别见到β葡萄糖苷酶、西妥昔单抗、西妥昔单抗-β葡萄糖苷酶偶联物的清晰条带。 比色法测定结果显示， 西妥昔单抗-β-葡萄糖苷酶偶联物的酶活性低于β-葡萄糖苷酶(U/L： 672.97±46.19 vs 869.50±57.28， t=5.972， P < 0.05)， 但仍保持良好的酶活性。 在荧光显微镜下可见到与偶联物共同作用的人膀胱癌 EJ 细胞带有红色荧光。 结论 Sulfo-SMCC 可成功将西妥昔单抗、β-葡萄糖苷酶偶联， 且偶联物仍能保持良好的酶活性与抗体活性。

关键词: 西妥昔单抗, β-葡萄糖苷酶, 偶联物, 膀胱肿瘤, Sulfo-SMCC, 癌

Abstract:

[Abstract] Objective To prepare cetuximab-β-glucosidase conjugates and to identify its enzymatic activity and an? tibody activity. Methods Cetuximab and β-glucosidase were crosslinked by Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (Sulfo-SMCC). Cetuximab- β-glucosidase conjugates and its enzymatic and antibody activity were examined by non-reduced SDS-PAGE, colorimetry and indirect immunofluorescence assay. Results We can see clear bands of β-glucosidase, cetuximab, cetuximab-β-glucosidase conjugates through electropherogram. Although the en? zymatic activity of cetuximab-β-glucosidase conjugates was lower than that of β-glucosidase (U/L： 672.97±46.19 vs 869.50± 57.28， t=5.972， P < 0.05) shown by colorimetry assay, it still maintain good enzymatic activity. Under fluorescence micro? scope, we can see the conjugates interacted with human bladder cancer EJ cells are in a red fluorescence. Conclusion Ce? tuximab, β-glucosidase were crosslinked successfully by Sulfo-SMCC without altered its enzymatic and antibody activity.

Key words: Cetuximab, β-glucosidase, Conjugates, urinary bladder neoplasms, Sulfo-SMCC, carcinoma