人CD244基因转染细胞株的构建及其单克隆抗体的研制

发布时间：2018-03-15 00:02

本文选题：人CD244　切入点：基因转染　出处：《苏州大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分人CD244基因克隆及其基因转染细胞株的建立【目的】构建稳定表达人CD244基因转染细胞株。【方法】通过RT-PCR获得人CD244全长cDNA，而后进行PCR扩增；将人CD244全长cDNA重组入逆转录病毒表达载体pEGZ-Term。经测序正确后，通过脂质体法将重组逆转录病毒载体pEGZ-Term/CD244和辅助病毒载体共转染包装细胞293T；收集含有完整重组逆转录病毒颗粒的293T细胞培养上清，用以感染L929细胞和BaF细胞；采用Zeocin筛选，获得Zeocin抗性的基因转染细胞，，并通过流式细胞术检测Zeocin抗性的基因转染细胞GFP和CD244的表达。【结果】成功克隆人CD244全长cDNA，并成功构建稳定表达人CD244的基因转染细胞株L929/CD244和BaF/CD244。【结论】本研究成功构建了稳定表达人CD244的基因转染细胞株，为研制鼠抗人CD244单克隆抗体和研究CD244生物学功能奠定了基础。第二部分鼠抗人CD244单克隆抗体的研制【目的】研制鼠抗人CD244单克隆抗体。【方法】以稳定表达人CD244的基因转染细胞株L929/CD244为免疫原，免疫6~8周龄的BALB/c小鼠；采用B淋巴细胞杂交瘤技术，将免疫小鼠的脾脏细胞与骨髓瘤细胞SP2/0进行细胞融合，并于HAT选择性培养基中培养；以L929/CD244作为阳性筛选细胞株，并以转染pEGZ-Term空载体的L929/mock细胞株和转染重组逆转录病毒载体pEGZ-Term/CD48的L929/CD48细胞株作为阴性对照，采用流式细胞术对杂交瘤细胞进行筛选；对所得阳性杂交瘤细胞进行多次克隆化培养，直至获得持续稳定分泌特异性鼠抗人CD244单克隆抗体的杂交瘤细胞株；采用快速定性试纸法鉴定单抗亚型；采用间接免疫荧光法检测单抗特异性，并分析所获单抗对健康人PBMC中T细胞和不同来源的肿瘤细胞表面CD244分子的识别。【结果】获得l株持续稳定分泌鼠抗人CD244单克隆抗体的杂交瘤细胞株，命名为21F8。经快速定性试纸分析鉴定，21F8的重链为IgM，轻链为κ链；腹水形成阳性率为100%，腹水的收获量平均为8ml/只。间接免疫荧光分析结果显示：单抗21F8能够特异性识别人CD244分子，并且能够识别人白血病细胞株U937和健康人PBMC中T细胞表面的CD244分子。【结论】成功获得l株持续稳定分泌鼠抗人CD244单克隆抗体的杂交瘤细胞株。
[Abstract]:The first part of Human CD244 Gene cloning and Establishment of Gene transfection Cell Line. [objective] to construct a stably expressed human CD244 gene transfected cell line. [methods] Human CD244 full-length cDNA was obtained by RT-PCR, and then amplified by PCR. The full-length cDNA of human CD244 was recombined into retrovirus expression vector pEGZ-Term.After sequencing, the recombinant plasmid pEGZ-Term. The recombinant retrovirus vector pEGZ-Term/CD244 and auxiliary virus vector were co-transfected into packaging cell 293T by liposome method. The culture supernatants of 293T cells containing intact recombinant retrovirus particles were collected to infect L929 cells and BaF cells. Zeocin resistant gene transfected cells were obtained and the expression of GFP and CD244 in Zeocin resistant gene transfected cells was detected by flow cytometry. [results] the full-length human CD244 cDNA was cloned successfully, and the transfected cell lines L929 / CD244 and Baf / CD244were successfully constructed. [conclusion] in this study, a stable expression of human CD244 gene transfected cell line was successfully constructed, which laid a foundation for the development of mouse anti-human CD244 monoclonal antibody and the study of the biological function of CD244. The second part: preparation of mouse monoclonal antibody against human CD244. [objective] to prepare mouse anti-human CD244 monoclonal antibody. [methods] Human CD244 gene transfected L929 / CD244 cell line was used as immunogen to immunize 68-week-old BALB/c mice, and the spleen cells of immunized mice were fused with myeloma cell SP2/0 by B lymphocyte hybridoma technique. L929 / CD244 was used as positive screening cell line, L929 / mock cell line transfected with empty pEGZ-Term vector and L929 / CD48 cell line transfected with recombinant retrovirus vector pEGZ-Term/CD48 as negative control. The hybridoma cells were screened by flow cytometry and the positive hybridoma cells were cloned and cultured for several times until the hybridoma cell lines secreting specific monoclonal antibodies against human CD244 were obtained. The monoclonal antibody subtype was identified by rapid qualitative assay, and the specificity of the monoclonal antibody was detected by indirect immunofluorescence assay, and the recognition of T cells and CD244 molecules on the surface of tumor cells from different sources in healthy human PBMC was analyzed. [results] A hybridoma cell line was obtained which continuously secreted murine anti-human CD244 monoclonal antibody and was named 21F8.The heavy chain of 21F8 was identified as IgMand the light chain was 魏 chain by rapid qualitative test paper analysis. The positive rate of ascites formation was 100 and the average yield of ascites was 8 ml / mouse. Indirect immunofluorescence analysis showed that McAb 21F8 could specifically recognize human CD244 molecule. And it can recognize the CD244 molecules on T cell surface in human leukemia cell line U937 and healthy human PBMC. [conclusion] A hybridoma cell line was successfully obtained which secreted murine anti-human CD244 monoclonal antibody stably and continuously.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】