抗IFITM1单克隆抗体的制备及检测

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EcoR 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1FITM1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、Westem-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。

Preparation and Application of A Monoclonal Antibody Against IFITM1

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 EcoR I 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 1FITM1-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 E. coli was successfully constructed. One hybridoma cell line secreting anti-IFITM 1 mAb 2F-1 belonged to IgG 1 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.