5种人IgG溶血活性及其作用机制的研究

发布时间：2018-09-04 12:17

【摘要】：免疫球蛋白G (immunoglobulin G, IgG)是人血清免疫球蛋白的主要成分,占总免疫球蛋白含量75%以上,是初级免疫应答中最持久、最重要的Ig,是唯一能通过胎盘的Ig,在主动、被动免疫中起重要作用。一般认为,非特异性免疫分子和特异性免疫分子之间不存在进化关系,但近年来学者们研究发现,众多无脊椎动物免疫分子不仅具有人Ig的典型功能域,而且具有明显的分子多态性,甚至具有部分获得性免疫学功能。由此提示,非特异性免疫分子和特异性免疫分子之间应该存在一定程度的进化关系,人IgG可能同样具备某些非特异性免疫学活性。本研究以健康人血清IgG为研究对象,采用亲和层析、糖组学、蛋白质组学、分子生物学的研究技术,深入探索IgG的溶血活性及其作用机制,所获主要研究结果如下： 首先,采用硫酸铵分级沉淀、纤维素DE 52离子交换层析和亲和层析从健康人血清分离5种人IgG:IgGg、IgGs-、IgGs+、IgGt-和IgGt+,其中纤维素DE 52离子交换层析纯化的IgG命名为IgGg;兔抗血蓝蛋白小亚基抗体亲和层析非特异性洗脱和特异性洗脱所得IgG分别命名为IgGs-和IgGs+;兔抗血蓝蛋白总蛋白抗体亲和层析非特异性洗脱和特异性洗脱所得IgG分别命名为IgGt和IgGt+。1-DE、2-DE和Western-blotting进一步分析表明,该5种蛋白确为人IgG,其中IgGg、IgGs+和IgGt+等3种IgG 2-DE图谱存在较大差异,与IgGg相比,IgGs+和IgGt+分别新增了1、5个蛋白质点。 其次,采用苯酚硫酸法、凝集素印迹比较5种人IgG糖基化的差异。结果发现,5种人IgG总糖含量存在明显的差异,其中IgGt+糖含量最高,为82.92μg/mg, IgGs-糖含量最低,为18.44μg/mg,前者约为后者的4倍。进一步研究显示,5种人IgG甘露糖含量亦存在明显差异,其中IgGt+甘露糖含量最高,IgGs-甘露糖含量最低,与总糖含量测定结果一致。由此说明,5种人IgG不仅存在蛋白多态性差异,而且存在糖基化差异,提示其可能同样存在免疫学活性的差异。 最后,运用溶血实验、1-DE、immunoblotting、渗透保护实验等技术,对比分析5种人IgG溶血活性的异同,并探索其可能的溶血机制。结果发现： 1) 5种人IgG对鸡、鼠、兔、人等7种红细胞的溶血活性存在极显著性差异,其中IgGs+和IgGt+2种IgG对7种红细胞均表现出明显的溶血活性,溶血率为30.99±0.01～100±0.08%,而IgGg、IgGs-和IgGt-等3种IgG对这7种红细胞几乎无溶血活性。 2) IgGs+和IgGt+分别经胰蛋白酶处理或高碘酸盐氧化后其溶血活性消失,表明人IgG溶血活性的发挥既与其蛋白构象有关,也与其糖基化程度高低有关。 3)进一步采用SDS-PAGE和Immunoblotting分析IgGt+与红细胞的相互作用,结果显示,与用双蒸水处理的红细胞相比,IgGt+处理的红细胞膜电泳后除了有IgGt+重链和轻链(55 kDa、26 kDa)条带,还新增一条分子量为121 kDa的高分子量条带,该条带亦能与抗人IgG抗血清发生特异性结合。采用胶内蛋白回收法纯化该高分子量蛋白,并进行SDS-PAGE和Immunoblotting分析,结果发现,121 kDa蛋白电泳后分为66.2 kDa和55 kDa两条带,且55 kDa蛋白条带能与抗人IgG抗体发生特异性结合。这些结果表明,人IgG溶血活性的发挥是通过其重链与红细胞膜紧密结合所致。 4)渗透保护实验发现,PEG可不同程度的阻断人IgG溶血活性的发挥,PEG4000,6000,8000对其阻断率分别为43.75±11.62%、60.41±1.27%和95.44±3.16%。由此可以认为,人IgG溶血作用机制可能为：人IgG分子重链通过与红细胞膜相互作用形成离子渗透孔,引起胶体渗漏而导致溶血。 综上所述,本课题首次研究发现人IgG具有非特异性免疫学活性—溶血活性,其溶血作用机制为胶体渗漏机制。所获研究结果对丰富人IgG的免疫学功能,探索脊椎动物适应性免疫的起源及其与无脊椎动物先天免疫的进化关系等具有重要意义。
[Abstract]:Immunoglobulin G (IgG) is the main component of human serum immunoglobulin, accounting for more than 75% of the total immunoglobulin content, is the most lasting primary immune response, the most important Ig, is the only Ig through the placenta, plays an important role in active, passive immunity. In recent years, many invertebrate immune molecules not only have typical domains of human Ig, but also have obvious molecular polymorphisms and even some acquired immune functions. This suggests that there should be some relationship between non-specific immune molecules and specific immune molecules. In this study, the hemolytic activity of human IgG and its mechanism were investigated by affinity chromatography, glycomics, proteomics and molecular biology.
Firstly, five kinds of human IgG: IgGg, IgGs-, IgGs +, IgGt - and IgGt +, which were purified by ammonium sulfate fractionation, cellulose DE 52 ion exchange chromatography and affinity chromatography, were separated from healthy human serum. The IgG purified by cellulose DE 52 ion exchange chromatography was named IgGg, and the rabbit anti-hemocyanin antibody was eluted by affinity chromatography and specific elution. IgG was named IgGs-and IgGs+, respectively; IgG was named IgGt and IgGt+.1-DE, 2-DE and Western-blotting respectively by affinity chromatography and specific elution of rabbit anti-haemocyanin total protein antibody. Further analysis showed that the five proteins were human IgG, and IgGg, IgGg, IgGs+and IgGt+were different from IgGg. IgGs+ and IgGt+ added 1,5 protein points respectively.
The results showed that the content of IgGt+sugar was the highest, 82.92 ug/mg, and the content of IgGs-sugar was the lowest, 18.44 ug/mg, the former was about four times of the latter. Further study showed that the content of mannose in five kinds of human IgG was also present. The results showed that IgGt + mannose content was the highest, IgGs - mannose content was the lowest, which was consistent with the determination of total sugar content.
Finally, hemolysis test, 1-DE, immunoblotting and osmotic protection test were used to compare and analyze the hemolytic activity of five kinds of human IgG, and explore the possible hemolytic mechanism.
1) The hemolytic activity of five kinds of human IgG to seven kinds of red blood cells, such as chicken, mouse, rabbit and human, was significantly different. IgGs + and IgGt + showed obvious hemolytic activity to seven kinds of red blood cells. The hemolytic rate was 30.99
2) The hemolytic activity of IgGs + and IgGt + disappeared after trypsin treatment or periodate oxidation respectively, which indicated that the hemolytic activity of human IgG was not only related to its protein conformation, but also to its glycosylation degree.
3) SDS-PAGE and Immunoblotting were used to analyze the interaction between IgGt+ and erythrocytes. The results showed that compared with the red blood cells treated with double evaporation water, IgGt + treated erythrocyte membrane electrophoresis had not only IgGt + heavy chain and light chain (55 kDa, 26 kDa) bands, but also a new high molecular weight band with 121 kDa. This band could also resist erythrocytes. The high molecular weight protein was purified by intracolloidal protein recovery method and analyzed by SDS-PAGE and Immunoblotting. The results showed that the 121 kDa protein was divided into 66.2 kDa and 55 kDa bands after electrophoresis. The 55 kDa protein band could bind specifically to the anti-human IgG antibody. These results showed that the human IgG hemolytic activity was enhanced. The development of sex is due to the close combination of heavy chain and red cell membrane.
4) The results of osmotic protection showed that PEG could inhibit the hemolytic activity of human IgG in varying degrees. The blocking rates of PEG 4000,6000,8000 were 43.75+11.62%,60.41+1.27% and 95.44+3.16% respectively. Colloid leakage causes hemolysis.
To sum up, this study found for the first time that human IgG has non-specific immunological activity-hemolytic activity, and its hemolytic mechanism is colloidal leakage mechanism. The results of this study are important for enriching the immunological function of human IgG, exploring the origin of adaptive immunity in vertebrates and its evolutionary relationship with innate immunity in invertebrates. Significance.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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