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Preparation of recombinant polyclonal antibody against Apple chlorotic leaf spot virus and its efficiency
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Maryam Afrashtehg    , Davoud Koolivand * , Mohammad Hajizadeh , Nahid Masoudi |
| Professor Department of Plant Protection, Faculty of Agriculture University of Zanjan, Zanjan, Iran , Koolivand@znu.sc.ir |
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Abstract: (627 Views) |
Apple chlorotic leaf spot virus (ACLSV) is one of the most important viruses infecting fruit trees. Preparation antibodies, ELISA kit and, biosensors are one of the applicable strategies to rapid and efficient screen a number of viral samples. To aim, the recent study was conducted to raise recombinant polyclonal antibodies to rapid detection of Apple chlorotic leaf spot virus by expressing coat protein gene in the prokaryotic system. First, the pTG19-ACLSV-CP was transformed to E. coli DH5α, the replicated plasmids were extracted and double digested was optimized by restriction enzymes, then the released fragment (Coat protein gene) was cloned into pET28 as an expression vector. Next, the expression construct (pET28a-ACLSV-CP) was transformed into E. coli strain BL21 (DE3) to express the coat protein gene. After optimization of expression the transformed cells, a protein band an approximate size of 27 kDa (22 kDa for coat protein and about 5 kDa for histidine tags) was observed at four hours after induction. The recombinant protein was purified by native methods using Ni-NTA Agarose column, then purified proteins were injected into New Zealand female rabbits in four steps as antigen. IgGs were purified from serums raised from immunized rabbits using an IgG purification kit. The efficiency of purified IgGs was approved that a 1: 1000 concentration of recombinant anti-ACLSV-CP antibodies are efficient to detect the desired antigens. The results showed the raised antibodies have enough specificity to detect Apple chlorotic leaf spot virus in gardens and seedlings. |
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| Keywords: ACLSV, Plasmid, Protein Expression, Coat Protein Gene, Purification |
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Full-Text [PDF 832 kb]
(118 Downloads)
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Type of Study: Research |
Subject:
Microrganisms and Viruses
Received: 2023/02/26 | Accepted: 2023/05/26 | Published: 2023/06/16
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