《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (4): 881-887.doi: 10.16431/j.cnki.1671-7236.2018.04.006

• 生物技术 • 上一篇    下一篇

定量检测猪繁殖与呼吸综合征病毒游离受体夹心ELISA方法的建立与应用

夏文龙1, 吴植2, 郭长明2, 朱善元2, 余树培1, 张鑫宇1, 夏晓莉1, 孙怀昌1   

  1. 1. 扬州大学兽医学院, 江苏省重要动物疫病与人兽共患病协同创新中心, 扬州 225009;
    2. 江苏农牧科技职业学院, 江苏省兽用生物制药高技术研究重点实验室, 泰州 225300
  • 修回日期:2017-12-01 出版日期:2018-04-20 发布日期:2018-04-25
  • 通讯作者: 孙怀昌(1956-),男,江苏淮安人,博士,教授,研究方向:兽医微生物学与免疫学,E-mail:sunh@yzu.edu.cn E-mail:sunh@yzu.edu.cn
  • 作者简介:夏文龙(1989-),男,江苏盐城人,博士生,研究方向:动物传染病防治,E-mail:469525768@qq.com
  • 基金资助:

    江苏高校优势学科建设工程项目(PAPD);江苏省农业科技自主创新资金项目CX (14)2087;江苏省自然科学基金(BK20151576);江苏农牧科技职业学院重点支持项目(NSFPT201631)

Establishment and Application of Sandwich ELISA for Quantitative Detection of Porcine Reproductive and Respiratory Syndrome Virus Soluble Receptors

XIA Wenlong1, WU Zhi2, GUO Changming2, ZHU Shanyuan2, YU Shupei1, ZHANG Xinyu1, XIA Xiaoli1, SUN Huaichang1   

  1. 1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
    2. Jiangsu Key Laboratory for High-tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Animal Husbandry and Veterinary College, Taizhou 225300, China
  • Revised:2017-12-01 Online:2018-04-20 Published:2018-04-25

摘要:

为了建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)游离受体定量检测方法,本试验用表达猪唾液酸黏附素(Sn)游离受体的重组腺病毒rAd-Sn4D-Fc感染PK-15细胞,以细胞培养上清纯化的Sn4D-Fc游离受体作为标准抗原,鼠抗猪Sn免疫血清为一抗,生物素标记兔抗猪Sn多克隆抗体为二抗,建立定量检测Sn4D-Fc游离受体的双抗体夹心ELISA方法;用rAd-Sn4D-Fc注射仔猪,定期采集血清进行游离受体检测。结果显示,纯化Sn4D-Fc标准抗原的纯度为94.3%,能被Sn免疫血清识别;一抗的最佳工作浓度为5 μg/mL蛋白,二抗的最佳稀释度为1:4 000,夹心ELISA检测标准抗原的灵敏度为0.4 ng/mL,对照抗原无交叉反应;夹心ELISA能从重组腺病毒注射猪血清中检测到Sn4D-Fc游离受体,最高表达量为6.54 ng/mL,持续时间为15 d。研究结果表明,建立的双抗体夹心ELISA可用于PRRSV游离受体的体内外定量检测。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); 游离受体; 夹心ELISA; 定量检测

Abstract:

To establish a sandwich ELISA for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) soluble receptors,porcine sialoadhesin (Sn) soluble receptor Sn4D-Fc was purified from the recombinant adenovirus-transduced PK-15 cell and used as the standard antigen,mouse anti-porcine Sn serum was used as the first antibody and biotin-labeled rabbit anti-porcine Sn polyclonal antibody as the second antibody.The established sandwich ELISA was validated by detection of the soluble receptor in the serum samples of rAd-Sn4D-Fc-injected pigs.The results showed that the Sn4D-Fc standard antigen was purified to a purity of 94.3%,and was able to be recognized by anti-Sn serum.By using 5 μg/mL of the first antibody and 1:4 000 of second antibody,the detection limit of established sandwich ELISA was up to 0.4 ng/mL without cross reaction to control antigen.The Sn4D-Fc soluble receptor could be detected in the rAd-injected pigs with the highest expression level of 6.54 ng/mL and the longest duration of 15 d.These data suggested that the established sandwich ELISA was usable for quantitative detection of PRRSV soluble receptors in vitro and in vivo.

Key words: porcine reproductive and respiratory syndrome virus (PRRSV); soluble receptor; sandwich ELISA; quantitative detection

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