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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 2  |  Page : 265-273

Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line


Department of Microbiology and Unit of Immunology, Alneelain University Faculty of Medicine, Alneelain Medical Research Center, Khartoum, Sudan

Correspondence Address:
Elham Omer Mahgoub
Department of Microbiology and Unit of Immunology, Alneelain University, Faculty of Medicine, Alneelain Medical Research Center, Khartoum
Sudan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.136551

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Background: A functional  single-chain fragment variable (scFv) recognizing the  MCF-7 breast cancer carcinoma cell line was constructed from the C3A8 hybridoma using phage display technology. Aim of Study: This study was conducted to evaluate the binding activity of scFv antibody recognise MCF-7 breast cancer cells carcinoma, the scfv antibody constructed and expressed in Escherichia coli periplasmic. Materials and Methods: The scFv coding sequence was cloned in frame with the pIII phage coat protein. The signal sequence included in the C terminus directed the expression of the scFv in the Escherichia coli periplasm. Following several rounds of biopanning, colonies that expressed a scFv that recognized MCF-7 cells in Western blots, ELISAs, and flow cytometry test were isolated. Results: A 750-bp scFv gene was successfully isolated. Cloning and two rounds of biopanning isolated the candidate with the highest activity (clone B7), as screened by ELISA. Following poly-acrylamide gel electrophoresis (SDS-PAGE) of the purified product, a 32-kDa band was observed. A similar-sized band was observed following Western blot analysis with an E tag-specific antibody. Binding reactivity of scFv antibody with MCF cells was determined using indirect ELISA and compared with monoclonal antibodies' reactivity. Also, flow cytometry was useful in further characterization to the binding reactivity of scFv antibody with MCF-7 cells. Conclusions: The recombinant antibody technology used in this study is a rapid and effective approach that will aid in the development of the next generation of immunodiagnostic reagents.

Abstract in Chinese

MCF-7乳腺癌细胞株的大肠杆菌 外周血浆中单链变异片段的构建、表达和特征 摘要 背景:利用噬菌体展示技术的c3a8杂交瘤构建功能性单链可变片段(scFv)识别的MCF 7乳腺癌癌细胞株。 研究目的:本研究旨在评价识别MCF 7乳腺癌细胞癌的单链抗体的结合活性,单链抗体在大肠杆菌周质中构建和表达。 材料与方法:克隆III噬菌体外壳蛋白scFv编码序列,包括在C末端信号序列的在大肠杆菌周质中的单链抗体的表达。筛选表达识别MCF 7细胞的scFv克隆,在Western印迹,ELISA,流式细胞仪中检测。 结果:750 bp scFv基因被成功分离。筛选分离具有最高的活性克隆B7。聚丙烯酰胺凝胶电泳(SDS PAGE)纯化后的产物观察到的是一个32 kDa带。Western印迹观察到同样大小的蛋白。与MCF细胞单链抗体的结合反应,用间接ELISA测定,并与单克隆抗体的反应性的比较。同时,流式细胞仪在进一步的MCF 7细胞的单链抗体的结合反应的特征描述上也是有用的。 结论:在这项研究中使用的重组抗体技术是一种快速、有效的方法,将有助于开发下一代免疫诊断试剂。 关键词:间接ELISA,噬菌体展示技术,周质,单链变异片段



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