Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

被引：0
作者：
Bashir, Nemat Sokhandan [1 ]
Koolivand, Davood [2 ]
Behjatnia, Seyed Ali Akbar [3 ]
机构：
[1] Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz
[2] Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan
[3] Department of Plant Protection, Faculty of Agriculture, University of Shiraz, Shiraz
来源：
Biotechnology | 2015年 / 14卷 / 04期
基金：
美国国家科学基金会;
关键词：
Antibody; Coat protein; Expression; GFLV; Recombinant;
D O I：
10.3923/biotech.2015.173.180
中图分类号：
学科分类号：
摘要：
Full length GFLV Coat Protein (CP) gene of Grapevine fanleaf Virus (GFLV) as one of the most destructive viruses of grapevine around the world was amplified from a previously prepared clone Kh4-5-3 with a pair of newly-designed primers. The CP gene was inserted into bacterial expression vector pET-21a (+) and the construct (pET21aGFLV CP) was cloned in Escherichia coli strain Rosetta. Optimization of expression was done by induction with 0.25, 0.5, 1, 2 and 6 mM final concentration of IPTG each for 3, 4, 6 and 16 h. Induction with 1 mM IPTG for 4 h proved to be the most efficient. Expression of the CP was verified with SDS-PAGE, Western blotting and Dot Immunobinding Blot Assay (DIBA) by the use of commercially available anti-GFLV antibody. The expressed CP was purified as the denatured or native protein before using as the antigen for raising anti-GFLV CP antiserum in rabbits. Specificity and titration of the antiserum was determined by Plate-Trapped Antigen Enzyme-Linked Immune Sorbent Assay (PTA-ELISA). Efficiency of the anti-GFLV CP IgG purified from the antiserum was demonstrated in Western blotting and Double Antibody Sandwich (DAS)-ELISA. This is the first report on preparation of polyclonal antibodies against recombinant GFLV CP isolate from Iran and its application in the virus detection. © 2015 Asian Network for Scientific Information.
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页码：173 / 180
页数：7
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