人CD200基因克隆与真核表达载体的构建

发布时间：2018-04-16 21:00

本文选题：CD200 + 克隆　；参考：《吉林大学》2005年硕士论文

【摘要】：CD200 分子是近年新发现的免疫超家族新成员,2000 年,在HLDA7(7th Workshop And Conference On Human Leukocyte Differentiation Antigens)会议上被正式命名。CD200(以前被称为MOX2、MRC OX-2抗原)是一种具有免疫调节功能的分子,目前动物实验已经证实CD200 分子有减轻自身免疫性疾病症状、延长移植物存活时间、抑制前炎症因子引起的流产、打破肿瘤免疫耐受等重要作用,因其具有治疗潜能而备受青睐,它独特的表达方式和效应功能有着广泛的应用前景,目前CD200 分子已成为一个新的研究热点。本研究从经Con A 刺激后的正常人外周血白细胞中获得总RNA,逆转录PCR 反应获得大量人CD200 基因,将其插入pcDNA3 表达载体,构建了真核表达质粒pcDNA3-CD200。用电穿孔法将重组质粒转染入U937 细胞中,经G418 筛选并连续传代培养30 代以上,采用流式细胞术技术测定其表达情况。本研究结果表明:成功的克隆了人CD200 基因并构建了真核表达质粒pcDNA3-CD200;用电穿孔的方法转染了U937 细胞,经G418 筛选后,空白对照组细胞全部死亡,转染目的基因组及空质粒组的细胞存活,并连续传代培养30 代以上,用FITC 标记的鼠抗人CD200 单克隆抗体采用流式细胞术技术监测该重组质粒的表达活性。空质粒组CD200 表达阳性细胞的百分率与对照组计数结果相比无统计学差异,而转染CD200 基因组表达阳性细胞的百分率与空质粒组及对照组结果相比有显著差异(p㩳0.01),前者阳性细胞百分率明显高于后两者。说明CD200 重组质粒转染成功并具有稳定的表达功能,稳定高效表达人CD200 基因的U937 细胞株已建立。
[Abstract]:CD200 molecule is a newly discovered member of the immune superfamily in recent years. It was officially named. CD200 (formerly known as MOX2MRC OX-2 antigen) at the HLDA7(7th Workshop And Conference on Human Leukocyte Differentiation antigens is a kind of immunomodulatory molecule.At present, animal experiments have proved that CD200 molecules have the important effects of relieving autoimmune disease symptoms, prolonging graft survival time, inhibiting abortion caused by preinflammatory factors, breaking tumor immune tolerance and so on.Its unique expression mode and effect function have a wide application prospect, CD200 molecule has become a new research hotspot at present.In this study, total RNAs were obtained from peripheral blood leukocytes stimulated by Con A, a large number of human CD200 genes were obtained by reverse transcription PCR reaction, and inserted into pcDNA3 expression vector, and the eukaryotic expression plasmid pcDNA3-CD200 was constructed.The recombinant plasmid was transfected into U937 cells by electroporation. The recombinant plasmid was screened by G418 and cultured for more than 30 generations. The expression of recombinant plasmid was determined by flow cytometry.The results showed that human CD200 gene was cloned successfully and eukaryotic expression plasmid pcDNA3-CD200 was constructed, and U937 cells were transfected by electroporation.The cells transfected with the genome and empty plasmid group survived and were cultured for more than 30 generations. The expression activity of the recombinant plasmid was detected by flow cytometry with FITC labeled mouse monoclonal antibody against human CD200.There was no significant difference in the percentage of CD200 positive cells between the empty plasmid group and the control group.The percentage of positive cells transfected with CD200 genome was significantly different from that of blank plasmid group and control group. The percentage of positive cells in the former group was significantly higher than that in the latter two groups.The results showed that the CD200 recombinant plasmid was successfully transfected and had stable expression function. The U937 cell line expressing human CD200 gene stably and efficiently was established.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【引证文献】