NYD-SP15基因的原核表达、抗体的制备以及细胞发育相关基因LIMS E的克隆分析

发布时间：2018-04-04 02:52

本文选题：原核表达　切入点：质粒　出处：《南京医科大学》2007年硕士论文

【摘要】： 目的：第一部分：通过构建与增生性病变相关基因NYD-SP15的原核表达质粒，体外诱导NYD-SP15蛋白的表达，免疫小鼠获得抗体，并把获得的多克隆抗体运用于增生性玻璃体视网膜病变的免疫组化研究。第二部分：研究Lims基因在PVR发生过程中RPE细胞发育中的作用。方法：第一部分：应用PCR技术扩增NYD-SP15全长开放阅读框，构建PET28a-NYD-SP15载体，再将其转入表达菌株，IPT6诱导表达，Ni柱纯化，免疫BALB／C小鼠，Western-blot法检测纯化的蛋白和抗体，，取视网膜增殖膜临床标本，抗体行免疫组化染色。第二部分：应用巢式RT—PCR，以小鼠cDNA为模板，扩增Lims基因不同剪切子，构入PinPointTM Xa-1P质粒，测序鉴定。结果：第一部分：成功地构建了PET28a-NYD-SP15原核表达质粒，并获得了高效表达NYD-SP15的BL21菌株，表达的His标签融合蛋白，分子量为58KD左右，经免疫小鼠获得了抗NYD-SP15抗体。经Western-blot法分析，抗体为NYD-SP15特异性抗体。免疫组化染色显示，NYD-SP15基因表达于视网膜色素上皮细胞的胞浆，呈现棕色染色。第二部分：测序表明克隆了新的Lims基因变异剪切体Lims E，该变异剪切体编码区为1164 bp，编码387个氨基酸。结论：第一部分：构建的NYD-SP15基因的原核表达载体，体外高效表达蛋白，免疫小鼠获得抗NYD-SP15抗体，在视网膜增殖膜的标本中，存在着NYD-SP15基因的表达，这些将为眼部增殖性玻璃体视网膜病变(PVR)的研究及治疗奠定坚实的基础。第二部分：比较基因组学分析显示，成功地克隆了一新的小鼠Lims基因剪切子Lims E，为进一步研究Lims基因可能在RPE细胞发育中的功能打下了基础。
[Abstract]:Objective:The first part: by constructing prokaryotic expression plasmid of NYD-SP15 gene associated with proliferative lesions, the expression of NYD-SP15 protein was induced in vitro, and antibody was obtained by immunizing mice.The obtained polyclonal antibodies were used in immunohistochemical study of proliferative vitreoretinopathy.Part two: to study the role of Lims gene in the development of RPE cells during the development of PVR.Methods:The first part: the full-length open reading frame of NYD-SP15 was amplified by PCR technique, and the PET28a-NYD-SP15 vector was constructed, and then transferred into the expressed strain, which was induced to express Ni column. The purified protein and antibody were detected by Western-blot method in immunized BALB/C mice, and the clinical specimens of retinal proliferating membrane were obtained.The antibody was stained by immunohistochemistry.The second part: using nested RT-PCR, using mouse cDNA as template, amplifying different splices of Lims gene, constructing PinPointTM Xa-1P plasmid and sequencing.Results:In the first part, the prokaryotic expression plasmid of PET28a-NYD-SP15 was successfully constructed, and the BL21 strain expressing NYD-SP15 efficiently was obtained. The fusion protein of His label was expressed with molecular weight of about 58KD. The anti-#en4# antibody was obtained by immunizing mice.Western-blot analysis showed that the antibody was NYD-SP15 specific antibody.Immunohistochemical staining showed that NYD-SP15 gene was expressed in the cytoplasm of retinal pigment epithelial cells and showed brown staining.Part two: sequencing showed that a new variant of Lims gene, Lims E, was cloned. The coding region of the variant was 1164 BP, encoding 387 amino acids.Conclusion:The first part: the prokaryotic expression vector of NYD-SP15 gene was constructed, and the protein was highly expressed in vitro. The anti NYD-SP15 antibody was obtained by immunizing mice. The expression of NYD-SP15 gene was found in the proliferative membrane of retina.These will lay a solid foundation for the study and treatment of ocular proliferative vitreoretinopathy (PVR).Part two: comparative genomics analysis showed that a new mouse Lims gene splitter Lims E was cloned successfully, which laid a foundation for further study on the function of Lims gene in the development of RPE cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R774.1;R346

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