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
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• epitopy genetika   metabolismus   MeSH
• genetické vektory genetika   MeSH
• geneticky modifikované rostliny MeSH
• imunizace MeSH
• klonování DNA MeSH
• lidé MeSH
• listy rostlin metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oligonukleotidy genetika   MeSH
• onkogenní proteiny virové metabolismus   MeSH
• protilátky virové krev   MeSH
• rekombinantní fúzní proteiny imunologie   metabolismus   MeSH
• tabák metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• virion imunologie   MeSH
• virové plášťové proteiny metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Human papillomavirus 16 (HPV16) E7 oncoprotein is a promising candidate for development of anti-cancer therapeutic vaccine. We have prepared the expression construct carrying mutagenized E7 oncoprotein fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein via 15 amino acids β-sheet linker. The fusion protein was expressed in Escherichia coli MC 1061 cells. We have obtained high level expression, but most of the protein remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular chaperones (TF, DnaK-DnaJ-GrpE, GroEL-GroES) were used. The immunological reactivity of expressed recombinant protein was evaluated with anti-E7 and anti-TMV antibodies. The distribution of expressed product during ultracentrifugation on sucrose gradient was studied.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• Escherichia coli genetika   MeSH
• exprese genu MeSH
• infekce papilomavirem virologie   MeSH
• klonování DNA metody   MeSH
• králíci MeSH
• lidé MeSH
• lidský papilomavirus 16 chemie   genetika   imunologie   MeSH
• molekulární chaperony chemie   genetika   MeSH
• mutageneze MeSH
• myši MeSH
• Papillomavirus E7 - proteiny chemie   genetika   imunologie   MeSH
• protilátky imunologie   MeSH
• rekombinantní fúzní proteiny chemie   genetika   imunologie   MeSH
• rozpustnost MeSH
• virové plášťové proteiny chemie   genetika   imunologie   MeSH
• virus tabákové mozaiky chemie   genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The E7 oncoprotein from Human papillomavirus type 16 (HPV16) is an attractive candidate for anti-cancer therapeutical vaccine development. In this study, we engineered different fusions of mutagenized coding sequence of E7 oncoprotein (E7ggg) with coat protein of Potato virus X (PVX CP) both on 5'- and 3'-terminus of PVX CP and evaluated the influence of the length of linker (no linker, 4, 15aa) connecting PVX CP and E7ggg on their production. At first the expression in Escherichia coli was conducted to assess the characteristics of the recombinant protein prior to be further produced in plants, that is, resultant proteins were used for screening of their immunological reactivity with antibodies against PVX CP and E7. Fusion proteins successfully expressed in bacteria and plants were partially purified and their reactivity and ability to form virus-like particles were evaluated with anti-E7 antibodies.

• MeSH
• 3' přiléhající oblast DNA MeSH
• 5' přiléhající oblast DNA MeSH
• Agrobacterium tumefaciens MeSH
• Escherichia coli MeSH
• exprese genu MeSH
• infekce papilomavirem imunologie   virologie   MeSH
• klonování DNA MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   imunologie   metabolismus   MeSH
• nádory děložního čípku imunologie   virologie   MeSH
• Papillomavirus E7 - proteiny genetika   imunologie   metabolismus   MeSH
• Potexvirus genetika   imunologie   metabolismus   MeSH
• proteinové inženýrství metody   MeSH
• protilátky imunologie   MeSH
• rekombinantní fúzní proteiny genetika   imunologie   metabolismus   MeSH
• tabák MeSH
• vakcíny proti papilomavirům chemie   genetika   MeSH
• virové plášťové proteiny genetika   imunologie   metabolismus   MeSH
• VLP vakcíny chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH