5種人IgG溶血活性及其作用機(jī)制的研究
發(fā)布時間：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,占總免疫球蛋白含量75%以上,是初級免疫應(yīng)答中最持久、最重要的Ig,是唯一能通過胎盤的Ig,在主動、被動免疫中起重要作用。一般認(rèn)為,非特異性免疫分子和特異性免疫分子之間不存在進(jìn)化關(guān)系,但近年來學(xué)者們研究發(fā)現(xiàn),眾多無脊椎動物免疫分子不僅具有人Ig的典型功能域,而且具有明顯的分子多態(tài)性,甚至具有部分獲得性免疫學(xué)功能。由此提示,非特異性免疫分子和特異性免疫分子之間應(yīng)該存在一定程度的進(jìn)化關(guān)系,人IgG可能同樣具備某些非特異性免疫學(xué)活性。本研究以健康人血清IgG為研究對象,采用親和層析、糖組學(xué)、蛋白質(zhì)組學(xué)、分子生物學(xué)的研究技術(shù),深入探索IgG的溶血活性及其作用機(jī)制,所獲主要研究結(jié)果如下： 首先,采用硫酸銨分級沉淀、纖維素DE 52離子交換層析和親和層析從健康人血清分離5種人IgG:IgGg、IgGs-、IgGs+、IgGt-和IgGt+,其中纖維素DE 52離子交換層析純化的IgG命名為IgGg;兔抗血藍(lán)蛋白小亞基抗體親和層析非特異性洗脫和特異性洗脫所得IgG分別命名為IgGs-和IgGs+;兔抗血藍(lán)蛋白總蛋白抗體親和層析非特異性洗脫和特異性洗脫所得IgG分別命名為IgGt和IgGt+。1-DE、2-DE和Western-blotting進(jìn)一步分析表明,該5種蛋白確為人IgG,其中IgGg、IgGs+和IgGt+等3種IgG 2-DE圖譜存在較大差異,與IgGg相比,IgGs+和IgGt+分別新增了1、5個蛋白質(zhì)點。 其次,采用苯酚硫酸法、凝集素印跡比較5種人IgG糖基化的差異。結(jié)果發(fā)現(xiàn),5種人IgG總糖含量存在明顯的差異,其中IgGt+糖含量最高,為82.92μg/mg, IgGs-糖含量最低,為18.44μg/mg,前者約為后者的4倍。進(jìn)一步研究顯示,5種人IgG甘露糖含量亦存在明顯差異,其中IgGt+甘露糖含量最高,IgGs-甘露糖含量最低,與總糖含量測定結(jié)果一致。由此說明,5種人IgG不僅存在蛋白多態(tài)性差異,而且存在糖基化差異,提示其可能同樣存在免疫學(xué)活性的差異。 最后,運用溶血實驗、1-DE、immunoblotting、滲透保護(hù)實驗等技術(shù),對比分析5種人IgG溶血活性的異同,并探索其可能的溶血機(jī)制。結(jié)果發(fā)現(xiàn)： 1) 5種人IgG對雞、鼠、兔、人等7種紅細(xì)胞的溶血活性存在極顯著性差異,其中IgGs+和IgGt+2種IgG對7種紅細(xì)胞均表現(xiàn)出明顯的溶血活性,溶血率為30.99±0.01～100±0.08%,而IgGg、IgGs-和IgGt-等3種IgG對這7種紅細(xì)胞幾乎無溶血活性。 2) IgGs+和IgGt+分別經(jīng)胰蛋白酶處理或高碘酸鹽氧化后其溶血活性消失,表明人IgG溶血活性的發(fā)揮既與其蛋白構(gòu)象有關(guān),也與其糖基化程度高低有關(guān)。 3)進(jìn)一步采用SDS-PAGE和Immunoblotting分析IgGt+與紅細(xì)胞的相互作用,結(jié)果顯示,與用雙蒸水處理的紅細(xì)胞相比,IgGt+處理的紅細(xì)胞膜電泳后除了有IgGt+重鏈和輕鏈(55 kDa、26 kDa)條帶,還新增一條分子量為121 kDa的高分子量條帶,該條帶亦能與抗人IgG抗血清發(fā)生特異性結(jié)合。采用膠內(nèi)蛋白回收法純化該高分子量蛋白,并進(jìn)行SDS-PAGE和Immunoblotting分析,結(jié)果發(fā)現(xiàn),121 kDa蛋白電泳后分為66.2 kDa和55 kDa兩條帶,且55 kDa蛋白條帶能與抗人IgG抗體發(fā)生特異性結(jié)合。這些結(jié)果表明,人IgG溶血活性的發(fā)揮是通過其重鏈與紅細(xì)胞膜緊密結(jié)合所致。 4)滲透保護(hù)實驗發(fā)現(xiàn),PEG可不同程度的阻斷人IgG溶血活性的發(fā)揮,PEG4000,6000,8000對其阻斷率分別為43.75±11.62%、60.41±1.27%和95.44±3.16%。由此可以認(rèn)為,人IgG溶血作用機(jī)制可能為：人IgG分子重鏈通過與紅細(xì)胞膜相互作用形成離子滲透孔,引起膠體滲漏而導(dǎo)致溶血。 綜上所述,本課題首次研究發(fā)現(xiàn)人IgG具有非特異性免疫學(xué)活性—溶血活性,其溶血作用機(jī)制為膠體滲漏機(jī)制。所獲研究結(jié)果對豐富人IgG的免疫學(xué)功能,探索脊椎動物適應(yīng)性免疫的起源及其與無脊椎動物先天免疫的進(jìn)化關(guān)系等具有重要意義。
[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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：2222077
[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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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