摘要
目的制备抗人ARMET(arginine-rich,mutated in early stage of tumors)的单克隆抗体(mAb),并鉴定其特性。方法将pET28a-ARMET转化到大肠杆菌BL21中,然后用IPTG诱导表达,用预装好的Ni-beads柱进行纯化,通过SDS-PAGE电泳及考玛斯亮蓝染色方法判断融合蛋白的表达量及纯度。将纯化的ARMET免疫雌性BALB/c小鼠后采用淋巴细胞杂交瘤技术,获取分泌抗ARMET杂交瘤细胞株。体内诱生腹水法制备mAb,间接ELISA法测定其效价及抗体亚型,辛酸-硫酸铵沉淀法及亲和层析法纯化mAb。用免疫荧光双标及Western blot法对抗体的特异性进行了鉴定。结果获得了纯度较高、浓度较高的融合蛋白;建立了7株稳定分泌特异性抗ARMET mAb的杂交瘤细胞株,诱生腹水法获得的抗体效价在1×10-5～1×10-7之间,且均有较好的特异性。结论已成功制备出抗ARMET mAb,为进一步用于临床诊断和实验研究奠定了基础。
Objective To prepare monoclonal antibodies （mAbs） against human ARMET（ arginine-rich, mutated in early stage of tumors）and characterize their properties. Methods pET28a-ARMET was transformed into E. coli BL21, and the fusion protein was expressed under IPTG induction. After purification, the protein ARMET was determined by SDS-PAGE and Commassie blue staining solution. Female BALB/c mice were immunized with purified ARMET protein. The mAb against ARMET was prepared by hybridoma technology. The mAbs were produced in mouse peritoneal cavity. Indirect ELISA was used to confirm the titrations and subtype of the monoclonal antibodies. The mAbs were purified with caprylic acid -ammonium sulfate precipitation （CA-AS） and affinity chromatography. Specificity was analyzed by immunofluorescenee and Western blot. Results We obtained a higher purity and con- centration of fusion protein and 7 hybridoma cell lines which could secreting specific anti-ARMET mAbs. The titers of 7 mAbs in ascitic fluid were 1 × 10^-5 ～ 1 × 10^-7. Conclusion The mAbs against ARMET have been prepared successfully, which provide useful reagent for further function research of ARMET.
出处
《安徽医科大学学报》
CAS
北大核心
2009年第6期665-669,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:30572219,30772557,30870881,230032)
安徽省科技攻关项目(编号:07010300191)
关键词
抗体
单克隆/分离和提纯
antibodies, monoclonal/isolationt ＆ purification
