人CD83转基因细胞的构建及鼠抗人CD83单克隆抗体的研制

发布时间：2018-06-09 06:00

本文选题：CD83 + sCD83　；参考：《苏州大学》2009年硕士论文

【摘要】： CD83是表达于成熟树突状细胞(DC)和活化的B、T淋巴细胞的重要功能分子,对T细胞在胸腺中的发育起着重要的作用。未成熟DC表面虽然缺少CD83的表达,但在其细胞内可检测到CD83蛋白。膜型CD83(membraneCD83,mCD83)蛋白可以水解形成可溶性CD83(solubleCD83,sCD83),存在于正常人血清中。sCD83能够影响DC的成熟,以及DC介导的T细胞增殖,具有免疫抑制活性。sCD83能够显著抑制实验性自身免疫病病变的发生,而且还能有效地抑制移植排斥反应。有研究认为与CD83相互作用的分子存在于单核和CD8+T细胞亚群。最近,一些研究发现了一些特殊的CD83+细胞,单核细胞在IFN-α作用下诱导成CD83+CD14+细胞群,该细胞群不具有典型DC的表型,却具有相似的吞噬功能,而单核细胞在GM-CSF作用下诱导成CD14lowCD83+ DCSIGN-细胞群,因此CD83的生物学特性及其信号传导途径尚待进一步研究。也已表明许多免疫分子的特异性抗体,可模拟或阻断该分子的信号。该类抗体可为深入研究免疫分子的生物学效应及分子机制提供重要的手段。同时通过特异抗体增强或减弱协同信号转导,对免疫功能低下或异常升高的疾病具有干预作用。因此,CD83分子转基因细胞的构建及功能性单克隆抗体的研制在基础研究及临床应用中具有重要价值。本论文共分为两个部分。 1.人CD83转基因细胞株的构建用TRIZOI从成熟的DC中提取总mRNA,逆转录成cDNA,以特异性引物进行扩增,然后进行双酶切装入pIRES2-EGFP载体,进行测序,测序正确后用脂质体转染L929细胞,筛选稳定表达株。pIRES2-EGFP载体中既有G418抗性标记基因,又有绿色荧光蛋白GFP报告基因,对于转基因细胞具有双重的筛选作用,大大提高了所筛细胞的阳性率。通过流式细胞仪反复筛选,获得一株稳定表达CD83的转基因细胞株,通过RT-PCR能够检测到目的基因的表达。 2.一株鼠抗人CD83单克隆抗体的研制及其特性鉴定以高表达膜型CD83分子的转基因细胞株L929/CD83为免疫原,采用B淋巴细胞融合技术,将免疫小鼠脾脏细胞与小鼠骨髓瘤细胞SP2/0进行融合。以人CD83基因转染小鼠成纤维细胞株L929/CD83及293/CD83为阳性筛选细胞,经反复筛选及克隆化培养,获得一株稳定分泌特异性鼠抗人CD83单抗的杂交瘤细胞株,命名为9D8。快速定性试纸法分析,9D8重链为IgG1,轻链为κ链。经体外连续传代培养,液氮冻存,复苏后生长良好,抗体分泌性能稳定。染色体数目分析显示,杂交瘤细胞株的染色体在100条以上。采用本室建立的腹水诱生法制备单抗,腹水形成阳性率约为80%以上,腹水的收获量平均为5 mL/只。经Protein G亲和层析柱分离纯化,腹水中抗体蛋白的得率为1.5~2.0 mg/mL。纯化抗体用于间接免疫荧光检测的用量为0.2~1μg/1x106细胞。经Western blot分析,9D8能与CD83分子特异性结合,表明9D8是CD83的特异性单抗。该单抗能识别人B淋巴瘤细胞株Daudi,骨髓瘤细胞株RPMI8226成熟的DC及活化的T淋巴细胞。总之,CD83编码区全长基因的成功克隆、重组载体的构建和稳定表达人CD83蛋白细胞株的建立为对该基因的进一步研究奠定了良好的基础。鉴于CD83在疾病诊断、免疫排斥和自身免疫性疾病中起着重要的作用,CD83转基因细胞的成功构建及鼠抗人CD83功能性单克隆抗体9D8成功研制为以后的研究工作打下了坚实的基础。
[Abstract]:CD83 is an important functional molecule expressed in mature dendritic cells (DC) and activated B, T lymphocytes. It plays an important role in the development of T cells in the thymus. Although the immature DC surface lacks the expression of CD83, the CD83 protein can be detected in its cells. The membrane type CD83 (membraneCD83, mCD83) protein can be hydrolyzed to form a soluble CD83. BleCD83, sCD83), the existence of.SCD83 in normal human serum can affect the maturation of DC and the proliferation of DC mediated T cells. The immunosuppressive activity.SCD83 can significantly inhibit the occurrence of experimental autoimmune disease and also effectively inhibit the graft rejection. The molecular interaction of CD83 is found to exist in mononuclear and C D8+T cell subsets. Recently, some special CD83+ cells have been found. Monocytes are induced into CD83+CD14+ cell groups under the action of IFN- alpha. The cell group does not have a typical DC phenotype, but has a similar phagocytic function, while monocyte is induced by GM-CSF to form a CD14lowCD83+ DCSIGN- cell group, so the biological special of CD83 is specific. Sex and its signal transduction pathways are still to be further studied.
It has also been shown that specific antibodies against many immune molecules can mimic or block the molecules' signals. This kind of antibody can provide an important means to further study the biological effects and molecular mechanisms of immune molecules. At the same time, it can interfere with diseases with low immune function or abnormal increase by enhancing or weakening the cooperative signal transduction by specific antibodies. Therefore, the construction of CD83 molecular transgenic cells and the development of functional monoclonal antibodies are of great value in basic research and clinical application. This paper is divided into two parts.
Construction of 1. CD83 transgenic cell lines
Using TRIZOI to extract total mRNA from mature DC, reverse transcriptase cDNA, amplify with specific primers, then carry out double enzyme cut into pIRES2-EGFP vector, sequence and transfect L929 cells with liposome after sequencing, and select both G418 resistance marker gene and GFP reporting basis of green fluorescent protein in stable expression strain.PIRES2-EGFP vector. Because of the double screening effect on the transgenic cells, the positive rate of the screened cells was greatly improved. A transgenic cell line with stable expression of CD83 was obtained through the flow cytometry, and the expression of the target gene could be detected by RT-PCR.
2. preparation and characterization of a mouse anti human CD83 monoclonal antibody
The transgenic cell line L929/CD83 with high expression of membrane CD83 molecule was used as immunogen, and B lymphocyte fusion technique was used to fuse the spleen cells of the mice with the murine myeloma cells SP2/0. The transfected mouse fibroblast cell line of human CD83 gene, L929/CD83 and 293/CD83 as positive screening cells, was screened and cloned by repeated screening and culturing. A hybridoma cell line that secretes the specific anti human CD83 monoclonal antibody, named 9D8. fast qualitative test paper method, 9D8 heavy chain IgG1, light chain kappa chain. After continuous subculture in vitro, liquid nitrogen is frozen, after resuscitation, the antibody secretion performance is stable. Chromosome number analysis shows that the chromosomes of hybridoma cell lines are 100 The positive rate of ascites was more than 80%, and the average harvest of ascites was 5 mL/, and purified by Protein G affinity chromatography column. The yield of antibody protein in ascites was 1.5~2.0 mg/mL. purified antibody used for indirect immunofluorescence detection using 0.2~1 mu g/1x106 cells. RN blot analysis shows that 9D8 can specifically bind to CD83 molecules, indicating that 9D8 is a specific monoclonal antibody to CD83. This monoclonal antibody can identify human B lymphoma cell line Daudi, myeloma cell line RPMI8226 mature DC and activated T lymphocyte.
In conclusion, the successful cloning of the full-length gene in the CD83 coding region, the construction of the recombinant vector and the establishment of a stable expression of the human CD83 protein cell line lay a good foundation for further research on this gene. In view of the important role of CD83 in the diagnosis of disease, immune rejection and autoimmune diseases, the successful construction of CD83 transgenic cells and the successful construction of the transgenic cells, The successful development of mouse anti human CD83 functional monoclonal antibody 9D8 laid a solid foundation for future research.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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