应用重组MSG1蛋白检测猪附红细胞体抗体的ELISA检测方法的建立

doi:10.3864/j.issn.0578-1752.2011.21.020

摘要/Abstract

摘要： 【目的】针对猪附红细胞体g1基因编码的MSG1表面粘附蛋白（rMSG1）和以此蛋白制备的多克隆抗体，建立相应的ELISA方法进行猪附红细胞体的临床检测，并进行效果评估，以获得一种猪附红细胞体感染的有效检测手段。【方法】将MSG1-pET28c_E.coli BL21菌株，在适宜的条件进行诱导表达，经菌体破碎、层析等步骤获取纯化的目的蛋白。以纯化的rMSG1制备免疫原接种小鼠，获取符合要求的多克隆抗体。以rMSG1和多克隆抗体建立ELISA检测方法，分析其敏感性、特异性、符合性和重复性。【结果】接种后获得了符合效价的抗血清；基于rMSG1的间接ELISA方法临床检测猪附红细胞体感染率为46.25%，其中阳性样本对已知小鼠抗血清有明显的阻断作用，并且与其它一些抗原无交叉反应；此ELISA方法的特异性和敏感性分别可达到97.06%和95.92%，较间接血凝试验(IHA)有更高的检出率。【结论】结果显示MSG1具有良好的抗原性，建立的ELISA方法具有较高的敏感性、特异性、符合性和可重复性，在临床检测中可以取得理想的效果。

关键词: 猪附红细胞体, MSG1, ELISA, 建立

Abstract: 【Objective】 An enzyme-linked immunosorbent assay (ELISA) system based on the polyclonal antibody against the adhesion protein MSG1 which was coded by the g1 gene of Mycoplasma suis(M. suis) was established, and was assessed as an effective method for detection of M. suis in swine. 【Method】 The MSG1-pET28c_E.coli BL21 was induced to express the MSG1protein of M. suis, and broken by ultrasound, the aim protein was purified by nickel affinity chromatography. The immunogen that was made of this purified recombinant protein was vaccinated to the mice to produce the polyclonal antibody. The polyclonal antibody and the recombinant protein were used to build an ELISA to detect M. suis, and the sensitivity, specificity, coincidence and repeatability of this method was analyzed, respectively. 【Result】 The polyclonal antibody was obtained whose titer had reached an adequate level. The infection rate of M. suis was 46.25% through the built ELISA method, the positive samples of them could hinder the polyclonal antibody from integrating with the MSG1protein markedly and didn’t react with some other antigens. The sensitivity and specificity values reached 95.92% and 97.06%, respectively, and a higher detection rate was confirmed through the ELISA method comparing with the IHA method. 【Conclusion】 The results show that the antigenicity of the protein MSG1 was satisfactory. This ELISA system had a higher sensitivity, specificity, coincidence and stability for the serodiagnosis of M. suis. A desirable result would be obtained from the detection of M. suis.

Key words: Mycoplasma suis, MSG1, ELISA, establishment

刘永夏, 刘建柱, 成子强, 周栋, 王振勇, 刘海涛, 王林, 柴同杰. 应用重组MSG1蛋白检测猪附红细胞体抗体的ELISA检测方法的建立[J]. 中国农业科学, 2011, 44(21): 4508-4515.

LIU Yong-Xia, LIU Jian-Zhu, CHENG Zi-Qiang, ZHOU Dong, WANG Zhen-Yong, LIU Hai-Tao, WANG Lin, CHAI Tong-Jie. Establishment of ELISA Based on Recombinant Protein MSG1 for the Detection of Mycoplasma suis Antibody[J]. Scientia Agricultura Sinica, 2011, 44(21): 4508-4515.

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