副猪嗜血杆菌外膜蛋白P2的提取及鉴定

中国畜牧兽医 ›› 2014, Vol. 41 ›› Issue (9): 225-230.

副猪嗜血杆菌外膜蛋白P2的提取及鉴定

罗银珠¹, 徐成刚¹, 周素明², 冯赛祥¹, 贺现辉¹, 樊惠英¹, 廖明¹, 辛朝安¹

1. 华南农业大学兽医学院, 农业部兽用疫苗创制重点实验室, 广东广州 510642;
2. 宁波大学海洋科学院, 海洋生物应用重点实验室, 浙江宁波 315211

收稿日期:2014-03-31 出版日期:2014-09-20 发布日期:2014-09-24
通讯作者: 廖明 E-mail:mliao@scau.edu.cn
作者简介:罗银珠（1983- ），女，广东人，硕士，研究方向：动物分子生物学。
基金资助:
农业部公益性行业科研专项经费项目（201303034）；农业科研杰出人才及其创新团队——现代农业人才支撑计划项目（农财发（2012）160号）；外膜蛋白P2在副猪嗜血杆菌诱导宿主细胞炎性反应中的作用及功能域研究（20114404110016）。

Extraction and Identification of Outer Membrane Protein P2 from Haemophilus parasuis

LUO Yin-zhu¹, XU Cheng-gang¹, ZHOU Su-ming², FENG Sai-xiang¹, HE Xian-hui¹, FAN Hui-ying¹, LIAO Ming¹, XIN Chao-an¹

1. Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Key Laboratory of Applied Marine Biotechnology, School of Marine Science, Ningbo University, Ningbo 315211, China

Received:2014-03-31 Online:2014-09-20 Published:2014-09-24

摘要/Abstract

摘要： 为简化副猪嗜血杆菌（Haemophilus parasuis，Hps）外膜蛋白中的单一蛋白外膜蛋白P2（OmpP2）的分离步骤，降低膜蛋白分离的设备要求，本试验用Hps标准3型（SW114）、5型（Nagasaki）菌株作为试验材料，选用溶菌酶、核酸酶对Hps进行酶解以获得其完整外膜，然后利用含有2% tritonX-100缓冲液对外膜进行进一步解离，最后利用适量浓度的胰酶对强疏水蛋白进行酶解，实现了Hps高纯度OmpP2的体外快速提取。SDS-PAGE结果显示，上述2株菌OmpP2大小分别约为43和38 ku；基质辅助激光解析电离飞行时间质谱（MALDI-TOF-TOF）鉴定结果显示，从上述蛋白酶解下来的肽段经比对与Hps的OmpP2完全吻合。斑点杂交（Dot-blot）显示采自Hps患病猪血清在点有5型菌株（Nagasaki）OmpP2和点有3型菌株（SW114）OmpP2 NC膜上均发生杂交显色反应，该结果暗示OmpP2是一个具有免疫活性的抗原。本研究为OmpP2功能及免疫保护性研究奠定了基础，也为副猪嗜血杆菌病免疫学诊断提供新的参考。

关键词: 副猪嗜血杆菌; 外膜蛋白P2; 提取; 鉴定

Abstract: In order to simplify theseparation steps of a single protein in the outer membrane protein P2 (OmpP2) of Haemophilus parasuis (Hps) and reduce the equipment requirements of membrane protein separation,Hps standard type 3 (SW114), 5 (Nagasaki) strains were used as experimental materials in this experiment.First, lysozyme, nucleic acid were used to enzyme digest enzymatic hydrolysis the Hps for its complete outer membrane. And then, using buffer containing 2% triton-X-100 for further disintegrate. At last, used the appropriate concentration of pancreatic enzymes to digest the strong hydrophobic protein. Finally, a rapid extraction of the Hps high-purity OmpP2 in vitro was found. The result of sodium dodecyl sulfate-poly propylene amide gel electrophoresis method (SDS-PAGE) showed that it was very consistent with our expection: The two strains of Hps OmpP2 was about 43 and 38 ku, respectively.The protease off peptides matched with Hps OmpP2 perfectly in mass spectrum (MALDI-TOF-TOF). This experiment had established a rapid extraction method of Hps OmpP2. It reduced the equipmental requirement and simplified the separation step of a single protein OmpP2 from the membrane protein. Both type 3 (SW114) and 5 (Nagasaki) strains OmpP2 had been found hybridization chromogenic reaction with serum collected from sick pigs in Dot-blot. It simplified that OmpP2 was an immunological antigens. This study would be worthy of the function and immune protective research for Hps OmpP2 and offered a new reference for Hps immunological diagnosis.

Key words: Haemophilus parasuis; outer membrane protein P2; extraction; identification

中图分类号:

S852.61

罗银珠, 徐成刚, 周素明, 冯赛祥, 贺现辉, 樊惠英, 廖明, 辛朝安. 副猪嗜血杆菌外膜蛋白P2的提取及鉴定[J]. 中国畜牧兽医, 2014, 41(9): 225-230.

LUO Yin-zhu, XU Cheng-gang, ZHOU Su-ming, FENG Sai-xiang, HE Xian-hui, FAN Hui-ying, LIAO Ming, XIN Chao-an. Extraction and Identification of Outer Membrane Protein P2 from Haemophilus parasuis[J]. , 2014, 41(9): 225-230.

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