人CGT52TGT、GGC54GAC和GGA57GAA MBL基因突变体在哺乳细胞中的表达及产物分析

发布时间：2018-04-30 08:48

本文选题：甘露聚糖结合凝集素 + 野生型　；参考：《第一军医大学》2005年硕士论文

【摘要】：甘露聚糖结合凝集素(mannan-binding lectin,MBL)系哺乳动物C型凝集素超家族中胶凝素家族的成员,是主要由肝细胞分泌的血浆糖蛋白。作为天然免疫系统的重要成份,MBL是补体凝集素激活途径的启动因子。近年来,越来越多的研究表明,各种病原体的反复感染与MBL缺损密切相关。人MBL基因位于染色体10q11.2-q21。现已发现3个可导致血清(浆)MBL水平低下并进而引起调理吞噬缺损的MBL结构基因点突变:CGT52TGT、GGC54GAC和GGA57GAA,其编码产物的改变分别是Arg(精氨酸)→Cys(半胱氨酸)、Gly(甘氨酸)→Asp(天冬氨酸)及Gly(甘氨酸)→Glu(谷氨酸)。这些点突变为常染色体共显性遗传,其基因频率在不同种族人群中差异甚大,但总体来说极高,如第52位密码突变在所研究过的人群中相对较低(≤0.1),第54位密码突变在欧亚人群中有较高频率(0.11～0.14),而第57位密码突变在撒哈拉非洲人群中的频率很高(0.23～0.29)。显然,MBL缺损是迄今所发现的最常见的免疫缺损病。因此,阐明MBL基因突变引起免疫缺损的机理具有极其重要的意义。然而,目前尚不清楚MBL基因突变导致血清(浆)MBL水平低下的直接原因,究竟是分泌减少还是结构不稳定?为了深入研究MBL基因突变引起免疫缺损的分子机制,并进一步阐明MBL的结构与功能的关系,我们选择CGT52TGT、GGC54GAC和GGA57GAA MBL突变体基因作为研究对象,将其在哺乳动物细胞中表达,并对瞬时表达和稳定表达的蛋白产物进行了初步分析。一、抗人MBL抗原肽多克隆抗体的制备与鉴定在对天然MBL分子优势表位预测的基础上,合成B细胞识别表位15 mer短肽NH2-EPGQGLRGLQGPPGK-COOH,将其与载体蛋白KLH偶联后免疫新西兰大白兔。经ELISA法检测抗体滴度为1:128 000。采用抗原肽亲和层析纯化法获得抗人MBL抗原肽多克隆抗体MSA(MBL specific-epitope antibody),其
[Abstract]:In recent years , more and more studies have shown that repeated infection of various pathogens is closely related to the activation pathway of lectin lectin .

however , it is not clear that that direct cause of low level of serum ( plasma ) mbl caused by the mutation of mbl gene is whether the secretion is decrease or the structure is unstable ? in order to study the molecular mechanism of the immune deficiency caused by the gene mutation of the mbl , and further clarify the relationship between the structure and function of mbl , we select the CGT52TGT , GGC54GAC and GGA57GAA mbl mutant gene as the research object to express them in mammalian cells , and analyze the transient expression and the stable expression of the protein product .

Preparation and identification of anti - human IgG polyclonal antibody

On the basis of the prediction of the dominant epitope of the natural mannan , B - cell recognition epitope 15 mer short peptide NH2 - EPGQGLRGLQGPPGK - COOH was synthesized , which was conjugated with the carrier protein to immune New Zealand white rabbits . The antibody titer was 1 : 128,000 by ELISA .

【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【参考文献】