Expression and purification of mucin 16 and preparation and characterization of anti-mucin 16 monoclonal antibody

Abstract

作者简介: 杨赟( 1988 － ) ，男，河南濮阳人，硕士生 Tel: 0592-2188250; E-mail: [email protected] Corresponding author，E-mail: xtli@ xmu.edu.cn[中文文摘]目的:在原核生物中表达带有His标签的mucin 16N端重组蛋白(简称为His-mucin 16N),制备抗mucin 16的单克隆抗体(mAb)。方法:将mucin 16基因片段插入原核表达载体pET-32,在大肠杆菌中表达重组蛋白,用亲和纯化方法纯化后免疫BALB/c小鼠,并进行细胞融合。筛选可稳定分泌抗mucin 16抗体的阳性单克隆杂交瘤细胞株,用Westernblot、ELISA、免疫荧光和免疫组化等方法分析和鉴定抗mucin16的mAb。结果:表达并纯化了His-mucin 16N蛋白;筛选出几株可稳定分泌特异性抗人mucin 16 mAb的细胞株;挑选出效价高、特异性好的1株进行纯化。获得的抗mucin 16 mAb,可用于Western blot、ELISA、免疫组化、免疫荧光等检测,并鉴定该抗体亚型为IgG1。通过上述免疫学实验,分析了在不同肿瘤细胞中mucin 16的表达情况。结论:在原核生物中成功表达和纯化带His标签的mucin 16N重组蛋白,制备出具有高特异性的抗mucin16的mAb。[英文文摘]AIM: To generate monoclonal antibodies ( mAbs) against mucin 16 using purified recombinant protein of human mucin 16 N terminus with His tag ( His-mucin 16N) as the antigen． METHODS: Mucin 16 N terminus was cloned into a prokaryotic expression vector pET-32． Hismucin 16N was then expressed in E． coli and purified by the affinity chromotography． Cell fusion was performed after the BALB/c mice were immunized with the purified His-mucin 16N protein． We screened hybridoma cell strains producing mAbs against mucin 16． The specificity and titer of the antibodies were characterized with ELISA，Western blotting， immunofluorescent and immunohistochemical staining． RESULTS: The recombinant protein of His-mucin 16N was expressed and purified． A few hybridoma cell strains which could secrete specific mAbs against mucin 16 were obtained，and one anti-mucin 16 mAb with good specificity and high titer was selected and purified． The isotype of this anti-mucin 16 mAb was determined as IgG1，which indicated that this anti-mucin 16 mAb could be used for ELISA，Western blotting，immunofluorescent and immunohistochemical staining． The endogenous expression of mucin 16 in various cancer cell lines or tissues was also examined with this antimucin 16 antibody by Western blotting and other immunoassays．CONCLUSION: The recombinant protein of Hismucin 16N was expressed and purified successfully，with which we prepared anti-mucin 16 mAb with good specificity and high titer．福建省科技重点项目(2011Y0050); 厦门市科技计划创新项目(3502Z20123009