人呼吸道卡他莫拉菌快速检测胶体金试纸的研制

doi:10.13560/j.cnki.biotech.bull.1985.2018-0184

生物技术通报 ›› 2018, Vol. 34 ›› Issue (9): 184-189.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0184

人呼吸道卡他莫拉菌快速检测胶体金试纸的研制

黄莹琪¹, 张秋¹, 陶冶¹, 胡征^1,2,3, 张改平⁴, 张华山^1,2,3

1. 湖北工业大学生物工程与食品学院,武汉 430068;
2. 发酵工程教育部重点实验室（湖北工业大学）,武汉 430068;
3. 工业发酵湖北省协同创新中心,武汉 430068;
4. 河南农业大学,郑州 450002

收稿日期:2018-03-06 出版日期:2018-09-26 发布日期:2018-10-10
作者简介:黄莹琪,女,硕士研究生,研究方向：发酵工程;E-mail：282942940@qq.com
基金资助:
湖北华龙生物制药有限公司资助

Development of a Colloidal Gold Immunochromatographic Test Strip for the Detection of Moraxella catarrhalis

HUANG Ying-qi¹, ZHANG Qiu¹, TAO Ye¹, HU Zheng^1,2,3, ZHANG Gai-ping⁴, ZHANG Hua-shan^1,2,3

1. School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068;
2. Key Laboratory of Fermentation Engineering of Ministry of Education（Hubei University of Technology）,Wuhan 430068;
3. Hubei Collaborative Innovation Center for Industrial Fermentation,Wuhan 430068;
4. Henan Agricultural University,Zhengzhou 450002

Received:2018-03-06 Published:2018-09-26 Online:2018-10-10

摘要/Abstract

摘要： 旨在研制一种快速、简便检测呼吸道感染病人呼吸道中卡他莫拉菌的试纸,并建立其检测方法。经生物信息学分析找到UspA1胞外结构域单一线性表位UspA1Line,偶联血蓝蛋白KLH形成复合蛋白UspA1Line-KLH,并利用已构建的表达UspA1抗原的工程菌,表达纯化重组蛋白UspA1-His,免疫新西兰大白兔制备多克隆抗体,取血清后利用Protein A亲和层析和多肽亲和层析两步纯化抗体,得到纯度高,特异性强的多克隆抗体。采用柠檬酸三钠还原法制备40 nm胶体金颗粒,与多克隆抗体结合,形成双抗体夹心,达到快速检测卡他莫拉菌的目的。结果显示,建立的检测方法可在10 min内完成对样本的检测,特异性检出样本中卡他莫拉菌,与其他常见的呼吸道病原菌无交叉反应,试纸条在24℃保存具有良好的重复性和稳定性。成功研制了可快速检测卡他莫拉菌的胶体金试纸条。

关键词: 卡他莫拉菌, 单一线性表位, UspA1, 免疫层析法

Abstract: The objective of this work is to develop a colloidal gold immunochromatographic test strip for the expeditious detection of Moraxella catarrhalis in the respiratory tracts of infected patients,and then to establish the corresponding approach. By bioinformatics analysis,the linear antigenic epitope on UspA1 of Moraxella catarrhalis was discovered and named UspA1Line1. The UspA1Line1 protein then ligated with KLH to generate the UspA1Line1-KLH protein. The already-constructed engineering strain for expressing UspA1 was used to express and purify the recombinant UspA1-His protein,which then was used to immune New Zealand rabbits for preparing the polyclonal antibody. Then the serum from the rabbits was purified by two steps of Protein A and polypeptide affinity chromatography,and the polyclonal antibody with high-purity and strong-specificity was acquired. The 40 nm colloidal gold particles were prepared by trisodium citrate reduction method,which then bound with the polyclonal antibody to form a double antibody sandwich,thus the goal of rapidly detecting M. catarrhalis was achieved. As a result,by the established approach M. catarrhalis was specifically detected in 10 minutes,and no cross-reactivity with common pathogens in upper respiratory tract occurred. The strips may be stored at 24℃ and their repeatability and stability remain well. In sum,a colloidal gold immunochromatographic test strip to rapidly detect M. catarrhalis is successfully developed.

Key words: Moraxella catarrhalis, linear antigenic epitope, UspA1, immunochromatography

黄莹琪, 张秋, 陶冶, 胡征, 张改平, 张华山. 人呼吸道卡他莫拉菌快速检测胶体金试纸的研制[J]. 生物技术通报, 2018, 34(9): 184-189.

HUANG Ying-qi, ZHANG Qiu, TAO Ye, HU Zheng, ZHANG Gai-ping, ZHANG Hua-shan. Development of a Colloidal Gold Immunochromatographic Test Strip for the Detection of Moraxella catarrhalis[J]. Biotechnology Bulletin, 2018, 34(9): 184-189.

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人呼吸道卡他莫拉菌快速检测胶体金试纸的研制

Development of a Colloidal Gold Immunochromatographic Test Strip for the Detection of Moraxella catarrhalis

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