马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用

doi:10.7668/hbnxb.2017.01.007

华北农学报 ›› 2017, Vol. 32 ›› Issue (1): 41-46. doi: 10.7668/hbnxb.2017.01.007

所属专题： 薯类作物；

马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用

宋志成¹, 费新敏¹, 杨煜², 乔利仙¹, 王晶珊¹, 李广存³, 郭宝太¹

1. 青岛农业大学生命科学学院, 山东省植物生物技术高校重点实验室, 山东青岛 266109;
2. 山东省农业科学院蔬菜花卉研究所, 山东济南 250100;
3. 中国农业科学院蔬菜花卉研究所, 北京 100081

收稿日期:2016-10-09 出版日期:2017-02-28
通讯作者: 郭宝太(1963-),男,山东青州人,教授,博士,主要从事植物分子生物学与基因工程研究。
作者简介:宋志成(1989-),男,山东青州人,硕士,主要从事植物基因工程研究。
基金资助:
山东省农业产业体系薯类创新团队遗传育种岗位基金项目（SDAIT-10-022-03）；济南市国际科技合作计划项目（2014003054）

Preparation of Polyclonal Antibody Against Recombinant CP of Potato virus A and Its Application to DAS-ELISA Detection

SONG Zhicheng¹, FEI Xinmin¹, YANG Yu², QIAO Lixian¹, WANG Jingshan¹, LI Guangcun³, GUO Baotai¹

1. College of Life Science, Qingdao Agricultural University, Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao 266109, China;
2. Vegetable Research Institute of Shandong Academy of Agricultural Sciences, Jinan 250100, China;
3. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2016-10-09 Published:2017-02-28

摘要/Abstract

摘要： 利用重组CP作抗原制备出马铃薯A病毒的多克隆抗体，并将其用于DAS-ELISA检测。以pET22b（+）为起始载体，构建了PVA-CP基因的原核表达载体pET22b-ACP，重组菌BL21（pET22b- ACP）经IPTG诱导表达出了分子量为30 kDa的特异性重组CP。利用高纯度重组CP为抗原免疫家兔，制备出了效价为1：512 k的抗血清。以PVA重组CP为抗原，用间接ELISA与直接ELISA分别测定重组CP纯化抗体（IgG）及其碱性磷酸酶标记物（IgG-AP）的活性，用DAS-ELISA测定2种抗体的活性，目测及OD值测定结果表明3种ELISA测定反应都呈阳性。以PVA病毒阳性标准物为抗原，在上述3种ELISA测定中也呈阳性反应，重组CP多克隆抗体及其酶标抗体与PVA有较强的反应信号。利用重组CP制备的多克隆抗体及其酶标抗体达到了马铃薯A病毒DAS-ELISA检测的要求。

关键词: 马铃薯A病毒, 重组CP, 多克隆抗体, 酶标记抗体, DAS-ELISA检测

Abstract: The purpose was to prepare polyclonal antibody against recombinant CP of Potato virus A,and apply it to DAS-ELISA.Taking pET22b(+) as the initial vector, CP gene prokaryotic expression vector of Potato virus A (pET22b-ACP) was constructed,the recombinant strain BL21 (pET22b-ACP) was induced with IPTG and specific recombinant CP of 30 kDa was obtained.Antiserum with the titer of 1:512k was prepared by using high-purity recombinant CP as antigen to immune rabbits.Using PVA recombinant CP as antigen,visual inspection and OD value measurement results indicated that reactions were respectively positive in indirect ELISA assay with the purified antibody (IgG) against the recombinant CP,in direct ELISA assay with the alkaline phosphatase-conjugated IgG (IgG-AP),and in DAS-ELISA determination with IgG against the recombinant CP and its AP-conjugated IgG.When PVA positive standard substance was used as antigen,reactions were also positive respectively in the three kinds of ELISA detections mentioned above.The polyclonal antibody against PVA recombinant CP and its enzyme-labeled antibody could meet the requirements of DAS-ELISA detection of Potato virus A.

Key words: Potato virus A, Recombinant CP, Polyclonal antibody, Enzyme-conjugated antibody, DAS-ELISA detection

中图分类号:

宋志成, 费新敏, 杨煜, 乔利仙, 王晶珊, 李广存, 郭宝太. 马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用[J]. 华北农学报, 2017, 32(1): 41-46. doi: 10.7668/hbnxb.2017.01.007.

SONG Zhicheng, FEI Xinmin, YANG Yu, QIAO Lixian, WANG Jingshan, LI Guangcun, GUO Baotai. Preparation of Polyclonal Antibody Against Recombinant CP of Potato virus A and Its Application to DAS-ELISA Detection[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(1): 41-46. doi: 10.7668/hbnxb.2017.01.007.

http://www.hbnxb.net/CN/Y2017/V32/I1/41

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马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用

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