| 刘灏 林智琪 符方勇 周忠信 刘正军.抗IFITM1 单克隆抗体的制备及检测[J].,2014,14(9):1606-1610 |
| 抗IFITM1 单克隆抗体的制备及检测 |
| Preparation and Application ofA Monoclonal AntibodyAgainst IFITM1 |
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| DOI： |
| 中文关键词: 干扰素诱导的跨膜蛋白-1 结肠癌 单克隆抗体 |
| 英文关键词: Interferon-induced transmembrane protein 1 Colon cancer Monoclonal antibody |
| 基金项目:国家自然科学基金面上项目(81172383)；广东省自然科学基金资助博士启动项目(S2011040002946) |
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| 中文摘要: |
| 目的：制备抗干扰素诱导的跨膜蛋白-1(interferon-induced transmembrane protein 1, IFITM1)的单克隆抗体，为检测IFITM1
及进一步研究其在结肠肿瘤发生过程中的作用提供实验基础。方法：以结肠癌患者的癌组织为材料，提取总RNA，以RT-PCR扩
增得到IFITM1 cDNA 序列，经ECoRⅠ和HindⅢ双酶切后，克隆入pGEX-4T-3 进行原核表达并纯化得IFITM1-GST；以该融合蛋
白免疫BALB/c 小鼠，淋巴细胞杂交瘤法制备单克隆抗体；采用ELISA、Western-blot及免疫组织化学法以制备的抗体检测结肠癌
患者结肠癌组织中的IFITM1。结果：成功构建了IFITM1 原核表达载体，获得了IFITM1-GST 重组蛋白；制备得到了1 株抗
IFITM1 单克隆抗体，腹水ELISA 效价为1:30000，抗体亚类为IgG1，可用于ELISA、Western-blot及免疫组织化学法检测结肠癌患
者结肠癌组织中的IFITM1。结论：获得了1 株可用于ELISA、Western-blot及免疫组织化学法的抗IFITM1 单克隆抗体2F-1，为进
一步研究IFITM1在结肠肿瘤发生过程中的作用提供了实验基础。 |
| 英文摘要: |
| Objective:To prepare a monoclonal antibody (mAb) against IFITM1 and provide experimental basis for the detection
of IFITM1 and explore the role of IFITM1 in the development of colorectal cancer.Methods:Total RNA was extracted from colonic
cancer cells collected from a patient with colon cancer. RT-PCR was performed to obtain the cDNA of IFITM1. After double digested
with EcoRⅠ and Hind Ⅲ, the cDNA of IFITM1 was cloned into pGEX-4T-3 vector and transformed into E.coli BL21 cells for fusion
IFITM1-GST expression. For the generation of monoclonal antibodies against IFITM1, the BALB/c mice were immunized with
IFITM1-GST, and the hybridoma techniques were employed. Then, we determine whether the mAbs could be applied to ELISA,
Western-blot and immunohistochemistry for the detection of IFITM1 in the cancer tissues from colon cancer patients.Results:A
recombinant plasmid with target gene and expressed recombinant IFITM1-GST in was successfully constructed. One hybridoma
cell line secreting anti-IFITM1 mAb 2F-1 belonged to IgG1 was obtained. The ELISA titer of ascetic fluids was 3× 10-4. mAb 2F-1 could
be applied to Western-blot, ELISA and immunohistochemistry for the detection of IFITM1 in the cancer tissues from colon cancer
patients.Conclusion:A monoclonal antibody that could be applied to Western-blot for the detection of IFITM1 in the sera or the cancer
tissues from colon cancer patie nts was successfully generated, which provided a new approach for the study of IFITM1 functions in the
origination and development of colon cancer. |
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