滑液支原体WVU1853株免疫相关膜蛋白的初步分析

doi:10.11843/j.issn.0366-6964.2017.02.015

摘要/Abstract

摘要：

膜蛋白是病原菌膜结构的主要组分，其不仅在病原菌的生命活动中具有重要的生理功能，且部分膜蛋白还与病原菌的感染及免疫应答密切相关。因此，本研究旨在应用免疫蛋白质组学方法分离、筛选并鉴定出滑液支原体免疫相关膜蛋白，以期为滑液支原体感染和免疫机制的研究及相关疾病诊断和防治方法措施的建立奠定基础。笔者应用培养至对数生长中后期的滑液支原体（Mycoplasma synoviae，MS）WVU1853株全菌分别免疫鸡和新西兰兔，制备滑液支原体鸡多抗和兔多抗。提取滑液支原体WVU1853株的膜蛋白，应用二维电泳技术对其进行分离，继而应用所制多抗进行Western blot，筛选出免疫原性膜蛋白，并通过质谱分析进行鉴定。结果表明，在筛选的8个蛋白质点中，3个蛋白质点为延伸因子EF-Tu，其余蛋白质点分别是丙酮酸脱羧酶α亚基、β亚基、NADH氧化酶、组氨酰-tRNA合成酶和二氢硫辛酰胺脱氢酶。其中组氨酰-tRNA合成酶的质谱分析得分太低，尚需进一步验证，而丙酮酸脱羧酶α、β亚基与作为滑液支原体的免疫相关膜蛋白首次被证实。本研究筛选并初步鉴定出滑液支原体7种免疫相关膜蛋白，为深入研究目标蛋白质的生物学特性及新型亚单位疫苗和诊断试剂的研发奠定了基础。

关键词: 滑液支原体, 免疫蛋白质组学, 膜蛋白, 鉴定

Abstract:

As a major component of membrane structure, membrane proteins not only have important physiological functions in the life of pathogen, but some of them are closely related to pathogen infection and immune response. Therefore, the main aim of this study was to isolate, screen and identify unknown possible immunogenicity proteins of Mycoplasma synoviae (MS) using immunoproteomics approach, which will lay a foundation not only for the study of infection and immune mechanism of MS but also for the establishment of diagnosis and prevention methods of related diseases. Chickens and New Zealand rabbits were immunized with MS cultured to mid-to-late logarithmic growth phase, respectively. Then the antiserum against MS from the chicken and the rabbit were prepared. Based on these, the membrane proteins of MS were isolated and then separated by two-dimensional electrophoresis. Finally the immunogenicity membrane proteins were screened and identified by Western blot combined with mass spectrum. The results showed that among the eight screened protein spots, three protein spots were identified as elongation factor EF-Tu, the rest of protein spots were identified as pyruvate decarboxylase alpha subunit, beta subunit, NADH oxidase, histidyl-tRNA synthetase and dihydrolipoyl dehydrogenase, respectively. Among them, it was confirmed for the first time that the pyruvate decarboxylase alpha subunits and beta subunits were the immune-related membrane proteins of MS, whereas the histidyl-tRNA synthetase need to be confirmed by further studies because protein score and protein Score CI% were too low. Several immune-related membrane proteins of MS strain WUV1853 were identified, which will be useful for further study on the biological function of target protein and development of new vaccines and diagnostic reagents.

Key words: Mycoplasma synoviae, immunoproteomics, membrane protein, identification

中图分类号:

S852.62

包世俊，丁小琴，邢小勇，伏小平,薛慧文，温峰琴 . 滑液支原体WVU1853株免疫相关膜蛋白的初步分析[J]. 畜牧兽医学报, 2017, 48(2): 316-323.

BAO Shi-jun, DING Xiao-qin, XING Xiao-yong, FU Xiao-ping, XUE Hui-wen, WEN Feng-qin. Preliminarily Analysis of Immune-related Membrane Proteins from Mycoplasma synoviae WVU1853 Strain[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 316-323.

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