We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This vector system consists of three domestication vectors carrying three GB parts-the cauliflower mosaic virus (CaMV) 35S promoter with PVX upstream of the second subgenomic promoter of the PVX coat protein (PVX CP SGP), nopaline synthase (NOS) terminator with PVX downstream of the first PVX CP SGP and the gene of interest (GOI). The full-length PVX clone carrying the sequence encoding a green fluorescent protein (GFP) as GOI was incorporated into the binary GB vector in a one-step reaction of three GB parts using the four-nucleotide GB standard syntax. We investigated whether the obtained vector named GFP/pGBX enables systemic PVX infection and expression of GFP in Nicotiana benthamiana plants. We show that this GB-compatible vector system can be used for simple and efficient assembly of PVX-based expression constructs and that it meets the current need for interchange of standard biological parts used in different expression systems.

• Keywords
• GoldenBraid, Nicotiana benthamiana, PVX vector, Potato virus X, transient expression,
• MeSH
• Genetic Vectors genetics   MeSH
• Potexvirus * genetics   MeSH
• Plants MeSH
• Nicotiana MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Green Fluorescent Proteins MeSH

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108-120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2 108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2 108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2 108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2 108-120 epitope were found after both methods of vaccine delivery.

• MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Epitopes genetics   metabolism   MeSH
• Genetic Vectors genetics   MeSH
• Plants, Genetically Modified MeSH
• Immunization MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• Plant Leaves metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oligonucleotides genetics   MeSH
• Oncogene Proteins, Viral metabolism   MeSH
• Antibodies, Viral blood   MeSH
• Recombinant Fusion Proteins immunology   metabolism   MeSH
• Nicotiana metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Virion immunology   MeSH
• Capsid Proteins metabolism   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• coat protein, Potato virus X MeSH Browser
• Epitopes MeSH
• L2 protein, Human papillomavirus type 16 MeSH Browser
• Oligonucleotides MeSH
• Oncogene Proteins, Viral MeSH
• Antibodies, Viral MeSH
• Recombinant Fusion Proteins MeSH
• Capsid Proteins MeSH