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	Mucin16蛋白的表达纯化及单克隆抗体的制备与鉴定; Expression and purification of mucin 16 and preparation and characterization of anti-mucin 16 monoclonal antibody
	杨赟 ; 丁宇婷 ; 龚慧婷 ; 于占娇 ; 李晓彤
	2012
关键词	mucin 16 CA125 蛋白纯化 单克隆抗体 杂交瘤 mucin 16 CA125 protein purification monoclonal antibody hybridoma
英文摘要	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。; 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.His-mucin 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 anti-mucin 16 antibody by Western blotting and other immunoassays.CONCLUSION: The recombinant protein of His-mucin 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)
语种	zh_CN
内容类型	期刊论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/119124]
专题	生命科学－已发表论文
推荐引用方式 GB/T 7714	杨赟,丁宇婷,龚慧婷,等. Mucin16蛋白的表达纯化及单克隆抗体的制备与鉴定, Expression and purification of mucin 16 and preparation and characterization of anti-mucin 16 monoclonal antibody[J],2012.
APA	杨赟,丁宇婷,龚慧婷,于占娇,&李晓彤.(2012).Mucin16蛋白的表达纯化及单克隆抗体的制备与鉴定..
MLA	杨赟,et al."Mucin16蛋白的表达纯化及单克隆抗体的制备与鉴定".(2012).

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