Evaluation and detection of a novel biomarker for breast cancer

纤维蛋白原(Fibrinogen)是大量存在于血液中的凝结蛋白它是纤维蛋白的前体，也是血液粘稠度和血液凝结的重要影响因素。纤维蛋白原(Fibrinogen)α链C端的605-629氨基酸组成的分子量为2660Da的多肽片段，简称Fib-α[605-629]。近期有美国哈佛大学的研究者报道，利用质谱法进行蛋白质组学分析证明乳腺癌病人血液中的Fib-α[605-629]含量与正常人血液中的含量存在非常显著的差异，乳腺癌病人血液中的该多肽含量要低于正常人的指数，而乳腺癌病人手术后血液中该多肽含量可恢复正常水平。因此该研究建议Fib-α[605-629]多肽可成为乳腺癌诊断及预后恢复情况的一种新的标志...Fib-α[605-629] peptide is an enzymolysis product of Fibrinogen. Recent reports by researchers at Havard University in US demonstrated that the content of Fib-α[605-629] peptide in breast cancer patients is significantly different from normal people, which is much lower than that in normal population, and it could be reverted to normal levels after surgical extirpation of the tumor. The new studies...学位：理学硕士院系专业：生命科学学院_细胞生物学学号：2162010115245

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

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

Characterization of NSE monoclonal antibodies and establishment of a double-antibody sandwich ELISA assay

通讯作者，E-mail: xtli@ xmu.edu.cn 作者简介: 丁焕弟( 1985 年－ ) ，女，在读硕士，主要从事抗肿瘤单克 隆抗体及诊断试剂盒的研究，E-mail: dinghuandi1125 @163.com。[中文文摘]目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。[英文文摘]Objective: Preparation and characterization of monoclonal antibodies against NSE protein，and establishment of a double-antibody sandwich ELISA assay． Methods: BALB/c mice were immunized by using purified recombinant NSE，and monoclonal antibodies were generated by hydridoma technique． These antibodies were characterized with ELISA，Western blot，Immunofluorescent and Immunohistochemical staining． The isotypes of these antibodies were determined with an antibody isotyping kit． With Horseradish Peroxidase labelled NSE monoclonal antibody，we were able to establish a double-antibody sandwich ELISA to detect NSE protein． Results: Two positive hybridoma cell lines were selected for test，the titers of these two monoclonal antibodies could reach 4． 2 × 107 －6. 5 × 107，and their isotypes were IgG2b． Our NSE antibodies could detect not only endogernous NSE protein from cells，but also secreted NSE protein from cells in culture medium by Western blot，in addition，they could be used for immunofluorescent and immunohistochemical staining． The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8． 85 ng /ml． Conclusion: Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers，and we established a doubleantibody sandwich ELISA assay which could be used for clinical test in future．福建省科技重点项目(编号项目No.2011Y0050); 厦门市科技计划项目(No.3502Z20123009