鹅细小病毒感染雏鹅的免疫球蛋白IgG/IgM变异性研究

doi:10.7668/hbnxb.2015.01.008

摘要/Abstract

摘要： 为探究IgG/IgM蛋白的分子特征、理化特性及病理学相关致病机理,对鹅细小病毒感染雏鹅的免疫球蛋白Ig进行了分级分离、纯化及IgG/IgM蛋白质结构和功能等生化-分子生物学分析。以GPVYZ感染雏鹅,采用(NH₄)₂SO₄分级分离鹅血清Ig,将Sephadex G-100凝胶柱在Utro-gel ACA22 凝胶柱色谱工作站上串联,分步聚集纯化出鹅血清IgM/G进行SDS-PAGE和生化分析。结果显示,初级沉淀鹅血清Ig,在非还原条件下,感染组(RD)缺失6个蛋白主带(128,114,69,59,55,48 kDa);而在还原条件下,RD组缺失7个蛋白主带(139,128,119,113,97,94,61 kDa),新增加3个蛋白主带(64,65,36 kDa),RD组和对照组(CK)的IgM和IgG带分别增加10,7倍和1.5,2倍。表明鹅血清IgM、IgG单体的高度可变性造成了在形状构型上的多态性,其亚基条带的变化是机体抗病潜能的重要标志;纯化鹅血清IgM/G,在非还原条件下,RD组的IgG缺失150 kDa分子和重链(64 kDa), 而在还原条件下,鹅血清IgG显示重链(60~70 kDa)特征蛋白带,重链62 kDa特异蛋白带仅出现在CK组的IgM/G中,同时RD的IgG还缺失91 kDa分子、IgG(ΔFc)(43 kDa)、轻链(25 kDa)蛋白带。提示GPV感染与鹅血清Ig发生相互作用,IgG与GPV感染密切相关,62 kDa重链基因可能是GPV的易感基因。试验还表明,RD组的鹅血清Ig含量仅为CK组的37%,IgG含量较IgM高2.56倍,RD组IgM、IgG比活力显著高于CK组,提示GPV拟制鹅血清IgM、IgG基因的表达,促使病毒感染雏鹅。稳定性试验显示,酸碱稳定性:鹅血清IgG > IgM;热稳定性IgM > IgG,RD组的IgM、IgG对碱和温度较为敏感。提示随pH值、温度的不同,Ig同时发生许多反应,GPV感染增加了鹅血清Ig的碱和温度敏感性。

关键词: 雏鹅, 鹅细小病毒, 免疫球蛋白M/G, SDS-PAGE, 凝胶层析

Abstract: To conduct isolating and purifying of immunoglobulin IgG/IgM from goslings infected Goose parvovirus YZ strain(GPV-YZ) and make analysis of protein structure and function by biochemical and molecular biology, and research the characteristics of Ig and the physicochemical properties and pathogenic mechanism of IgG/IgM protein.Immunoglobulin(Ig) was isolated and purified from goslings infected Goose parvovirus(RD)and control group(CK)by ammonium sulphate fractionation chromatographies on Sephadex G-100 and Utro-gel ACA22.The results of SDS-PAGE and biochemical analysis showed that there were deleted 6 main protein(128, 114, 69, 59, 55, 48 kDa) band of the primary sedimentation Ig from serum of goslings infected Goose parvovirus in non reductive conditions, but under reductive conditions, deleted 7 main protein(139, 128, 119, 113, 97, 94, 61 kDa) band and appeared three new main protein(64, 65, 36 kDa) band of the primary sedimentation Ig from serum of goslings infected Goose parvovirus.There were increased respectively 10, 7 times or 1.5, 2 times of protein bands of IgM and IgG from goslings infected Goose parvovirus and control group, and indicated that the high variability macromolecular IgM/G monomers caused an abundant polymorphism in shape configuration, and the IgM/G subunit change was an important marker of the resistance potential in goslings.There were deleted 150 kDa band and heavy chain (64 kDa) of the purified IgG from serum of goslings infected Goose parvovirus in non reductive conditions, but under reductive conditions, the IgG showed heavy chain(60~70 kDa) characteristic protein band in goslings infected Goose parvovirus and control group.The 62 kDa specific heavy chain bands only appeared in the IgM/G of control group, and deleted the 91 kDa, IgG(ΔFc)(43 kDa)and light chain (25 kDa) protein band from serum of goslings infected Goose parvovirus.Those indicated that there existed the interaction between GPV infection and Ig.The IgG is associated with GPV infection, and 62 kDa heavy chain gene might be the susceptible gene of GPV.The results also indicated that the Ig content of goslings infected Goose parvovirus was only 37 percent of control group, and IgG content was 2.56 times higher than that of IgM in serum of goslings infected Goose parvovirus. The specific activity of IgM/G of goslings infected Goose parvovirus was significantly higher than the control group. Those indicated that the expression of immunoglobulin gene were reduced by goslings infected Goose parvovirus, and stimulated goslings infected Goose parvovirus.The specific activity of IgM/G of goslings infected Goose parvovirus was significantly higher than the control group.Those indicated that the expression of immunoglobulin gene were reduced by goslings infected Goose parvovirus, and stimulated goslings infected Goose parvovirus.The stability test showed that the IgG was pH stability better than IgM in goslings infected Goose parvovirus, but the IgM was better thermal stability than IgG in goslings infected Goose parvovirus and IgM/G were more sensitive to alkali and temperature in goslings infected Goose parvovirus.Those indicated that with temperature, pH different immunoglobulin might also occur many reactions, and the alkali and temperature sensitivity of Ig increased in goslings serum infected Goose parvovirus.

Key words: Goslings, Goose parvovirus, Immunoglobulin M/G, SDS-PAGE, Gel chromatography

中图分类号:

S853.5
Q78

朱新产, 邵艳红, 朱峰伟, 杨丽金. 鹅细小病毒感染雏鹅的免疫球蛋白IgG/IgM变异性研究[J]. 华北农学报, 2015, 30(1): 42-53. doi: 10.7668/hbnxb.2015.01.008.

ZHU Xin-chan, SHAO Yan-hong, ZHU Feng-wei, YANG Li-jin. Study on the Molecular Variability of Immunoglobulin IgG/IgM from Goslings Infected Goose Parvovirus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(1): 42-53. doi: 10.7668/hbnxb.2015.01.008.

http://www.hbnxb.net/CN/Y2015/V30/I1/42

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