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

Sokhandan, Nemat; Koolivand, Davoud; Behj, Seyed Ali Akbar

doi:10.3923/biotech.2015.173.180

Cited by 3 publications

(4 citation statements)

References 33 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To produce pBluescript II SK-GFLV-L2 construct, GFLV L2 gene was synthesized and cloned into the NcoI and BamHI sites by BioBasic Company (Toronto, Canada). pGEM-GFLV CP [52] and pBluescript II SK-GFLV-L2 were used as templates for amplification of GFLV CP and GFLV L2 genes using a primer set (Sense primer:5′-CCA GCC GGC GAT GGC CAT GGG ATT AGC TGG TAG AGG-3′; antisense primer: 5′-ACG GAG CTC GAA TTC GGA TCC CTA GAC TGG GAA ACT GGT TCT CCA-3′ which includes two homologous region CCA GCC GGC GAT GGC and ACG GAG CTC GAA TTC GGA TCC) and Phusion High Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). The amplified fragments were subcloned in the pET26 vector in frame with the pelB leader sequence using the Gibson assembling cloning master mix for 15 min at 50 °C (New England Biolabs) and transformed into E. coli DG1 (Eurogentec, Liege, Belgium).…”

Section: Methodsmentioning

confidence: 99%

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

et al. 2019

View full text Add to dashboard Cite

BackgroundVirus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated.ResultsThe epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure.ConclusionsThe results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

show abstract

Section: Methodsmentioning

confidence: 99%

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

et al. 2019

View full text Add to dashboard Cite

show abstract

“…The antiserum was titrated against CP42 in a plate-trapped antigen (PTA)-ELISA by the method elucidated by Sokhandan et al (2015) with modifications (16).…”

Section: Methodsmentioning

confidence: 99%

“…Polyclonal antibodies were recently prepared against GFLV CP expressed in the E. coli (16), however in the present study only 42kDa segment of the CP gene that was predicted to contain epitopes; a shortened segment more than 56 kDa of the whole protein, was synthesized and expressed in the form of inclusion body. Then, the inclusion body was used as the antigen.…”

Section: Introductionmentioning

confidence: 91%

“…Conventionally, virus particles are used as the viral antigen for production of polyclonal and monoclonal antibodies. Since virus purification is a very sensitive process and needs a very expensive equipment (14), this method has been replaced by recombinant DNA technology for production of the structural proteins such as viral CPs (15, 16). One advantage of this system is the production of the protein (antigen) in large quantities (17).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Production and Partial Purification of the Grapevine Fanleaf Virus Coat Protein 42 Polyclonal Antibody Against Inclusion Body Expressed in Escherichia coli

et al. 2018

View full text Add to dashboard Cite

Background Expression of virus coat protein (CP) in Escherichia coli often leads to production of partially folded aggregated proteins which are called inclusion bodies. Grapevine fanleaf virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran. Objective The main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV. Material and Methods An antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3). The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one. Results The expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody. Conclusions It was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.

show abstract

Immunodiagnosis of bunchy top viruses in abaca with polyclonal antibodies against their recombinant coat proteins

Koh

Zaulda

Barbosa

et al. 2020

Archives of Phytopathology and Plant Protection

View full text Add to dashboard Cite

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

Cited by 3 publications

References 33 publications

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

Production and Partial Purification of the Grapevine Fanleaf Virus Coat Protein 42 Polyclonal Antibody Against Inclusion Body Expressed in Escherichia coli

Immunodiagnosis of bunchy top viruses in abaca with polyclonal antibodies against their recombinant coat proteins

Contact Info

Product

Resources

About