Expression of rabies virus G protein in carrots (Daucus carota)

Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, María Teresa de Jesús; Gómez-Lim, Miguel Á.

doi:10.1007/s11248-009-9278-8

Cited by 38 publications

(14 citation statements)

References 41 publications

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“…As a transformation method biolistics was used. The average expression level in obtained transgenic plants was 25 µg/g of fresh tissue, equalizing about 1.4 % TSP, which is the same amount as was obtained in carrot by Anaya et al (2009). When orally immunized with 2 mg of obtained G protein one single time, sheep (the main victims of rabies infections in Latin America) elicited anti-rabies neutralizing serum Ab and a protective immunity against rabies virus infection with a survival rate of 83 % was obtained, the same as the efficiency obtained by inactivated commercial vaccine (Loza-Rubio et al, 2012).…”

Section: Rabiessupporting

confidence: 62%

“…The pioneering work in expressing G protein in transgenic plants was done in 1995 by McGarvey et al by expressing an unmodified G protein gene under the control of the CaMV 35S promoter in Agrobacterium-transformated tomato leaves and fruits. Since then, G protein has been expressed on its own in plants such as tobacco (Modelska et al, 1997;Yusibov et al, 1997;Ashraf et al, 2005,), spinach (Modelska et al, 1997), carrot (Anaya et al, 2009) and maize (Loza-Rubio et al, 2012). A full-length rabies virus nucleoprotein has also been expressed in tomato (Perea Arango et al, 2008), cholera toxin subunit B (CT-B)-rabies glycoprotein fusion protein has been expressed in tobacco (Roy et al, 2010) and tobacco seeds (Tiwari et al, 2009), chimeric peptide containing rabies G protein and N protein determinants was expressed in tobacco and spinach (Yusibov et al, 2002) and a rabies glycoprotein-ricin toxin B chain fusion protein has been expressed in tomato hairy roots (Sigh et al, 2015).…”

Section: Rabiesmentioning

confidence: 99%

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Production of vaccines for treatment of infectious diseases by transgenic plants

Ledl

Luthar

2016

AAS

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Since the first pathogen antigen was expressed in transgenic plants with the aim of producing edible vaccine in early 1990s, transgenic plants have become a well-established expression system for production of alternative vaccines against various human and animal infectious diseases. The main focus of plant expression systems in the last five years has been on improving expression of well-studied antigens such as porcine reproductive and respiratory syndrome (PRRSV), bovine viral diarrhea disease virus (BVDV), footh and mouth disease virus (FMDV), hepatitis B surface antigen (HBsAg), rabies G protein, rotavirus, Newcastle disease virus (NDV), Norwalk virus capsid protein (NVCP), avian influenza virus H5N1, Escherichia coli heat-labile enterotoxin subunit B (LT-B), cholera toxin B (CT-B), human immunodeficiency virus (HIV), artherosclerosis, ebola and anthrax. Significant increases in expression have been obtained using improved expression vectors, different plant species and transformation methods.Key words: transgenic plants; vaccine production; recombinant proteins; antibodies; infectious diseases; risk assessment IZVLEČEK ZDRAVLJENJE NALEZLJIVIH BOLEZNI S CEPIVI PRIDOBLJENIMI S TRANSGENIMI RASTLINAMIPridobivanje aktivnih sestavin za cepiva s transgenimi rastlinami se je začelo v devetdesetih letih prejšnjega stoletja. Od takrat se transgene rastline uveljavljajo kot alternativni ekspresijski sistem za sintezo aktivnih komponent za cepiva proti številnim humanim in živalskim nalezljivim boleznim. V zadnjih petih letih je bil glavni poudarek razvoja namenjen optimizaciji rastlinskih ekspresijskih sistemov za dobro proučene antigene, kot so virus prašičjega respiratornega in reproduktivnega sindroma, virus goveje virusne diareje, virus parkljevke, površinski antigen hepatitisa B, G protein virusa stekline, rotavirus, virus atipične kokošje kuge, plaščni protein Norwalk virusa, sev ptičje gripe H5N1, temperaturno nestabilna B podenota enterotoksina Escherischia coli, kolera toksin B, HIV, arterioskleroza, ebola in antraks. Uporaba optimiziranih transformacijskih metod in ekspresijskih vektorjev v kombinaciji s primerno izbrano rastlinsko vrsto je v večini naštetih primerov vodila do opaznega izboljšanja sinteze posameznih učinkovin.

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Section: Rabiessupporting

confidence: 62%

Section: Rabiesmentioning

confidence: 99%

Production of vaccines for treatment of infectious diseases by transgenic plants

Ledl

Luthar

2016

AAS

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“…Due to its large protein size, it would be very hard to express such a big protein in prokaryotic or eukaryotic system, which restricted the development of its antibody and research progress of L protein. By far amongst rabies virus proteins, G protein (27)(28)(29)(30)(31), nucleoprotein (32)(33), M protein (34) and phosphoprotein (35) have been successfully expressed in varieties of hosts and most often led to production of an antibody. However and until now, no commercial antibody for RABV L protein was developed to be introduced to the market.…”

Section: Discussionmentioning

confidence: 99%

Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

Zhang¹,

Jin²,

Sun³

et al. 2015

Iran J Biotech

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Rabies is an old known disease to mankind in recorded history. Approximately 55,000 individuals die from rabies, caused by rabies virus (RABV), worldwide per annum (1). The ~12 kb genome of RABV is composed of five genes that encode the structural proteins of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), matrix protein (M), and large protein (L) (2). The cDNA of L protein encodes a long polypeptide of 2128 amino acids with a relative molecular weight of 243.09 kDa (silver-haired bat-associated strain (SHBRV)). L protein (named after its large molecular weight) is considered to work with the phosphorylated non-catalytic subunit of viral RNA polymerase, P protein, and as a catalytic subunit of RNA polymerase (3). The polymerase complex of L and P displays all the enzymatic activity of transcription, including co-transcriptional modifications of RNAs, such as capping and polyadenylation, and the initiation and elongation of transcripts (4). L protein functions were mostly predicted from studies on vesicular stomatitis virus. In recent years, reports about RABV were mainly focused on the viral G (5-7), P (8-14), M (15-18), and N (9,19-24) proteins. However, the work on L protein of RABV has been relatively limited. Its potential role in the virus cycle and host factors interacting with L protein remains to be determined.In our previous study (9), we have established 11 mAbs, including three neutralizing antibodies, one anti-nucleoprotein antibody and seven mAbs against phosphoprotein, through a strategy of suckling mouse brain antigen immunization. The large molecular size of L protein and the lack of commercially available antibodies against RABV L protein restricted the progression of RABV L protein research work. Development of an effective antibody is especially Background: Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. Objectives: A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. Materials and Methods:The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET28a and transformed into E. coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. Results:The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brai...

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“…All three detergents were sourced from Sigma. The detergents, CHAPS, NP-40, and Triton X-100, were added at final concentrations of 1%, 0.2%, and 0.1%, respectively (1,19). The cell pellet, stored frozen at Ϫ20°C, was thawed at room temperature, and 1 ml of buffer-detergent solution per 2 ϫ 10 7 cells was added ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

Rajendran

Subramanian

Sivakumar

et al. 2011

Clin. Vaccine Immunol.

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Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membraneassociated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

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Expression of rabies virus G protein in carrots (Daucus carota)

Cited by 38 publications

References 41 publications

Production of vaccines for treatment of infectious diseases by transgenic plants

Production of vaccines for treatment of infectious diseases by transgenic plants

Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

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