- Publikační typ
- abstrakt z konference 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
- 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
The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Omega. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.
- MeSH
- ELISA MeSH
- králíci MeSH
- monoklonální protilátky biosyntéza MeSH
- nemoci rostlin virologie MeSH
- Potyvirus imunologie MeSH
- protilátky virové biosyntéza MeSH
- rekombinantní proteiny imunologie MeSH
- Solanum tuberosum virologie MeSH
- tabák virologie MeSH
- virové plášťové proteiny imunologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
Malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40, NADP-ME), which was found in chloroplasts, was isolated from tobacco leaves (Nicotiana tabacum L.) almost homogenous. The specific enzyme activity was 0.95 mmol min-1 mg-1. The enzyme pH optimum was found between pH 7.1 and 7.4. The affinity of NADP-ME to substrates (L-malate and NADP+) was evaluated in the presence of divalent metal ions (Mg2+, Mn2+, Co2+, Ni2+). The value of the apparent Michaelis constant of NADP-ME for L-malate was dependent on the ion cofactor, while no such relationship was found for NADP+. The dependence of the reaction rate on concentration of Mg2+ indicates the presence of more than one binding site for these ions in NADP-ME. Likewise, the sigmoidal dependence of the reaction rate on Mn2+ concentration and the value of Hill coefficient 7.5 indicate the positive cooperativity of the reaction kinetics in the presence of the ions. The effect of Co2+ and Ni2+ ions was analogous to that of Mn2+ ions; however, the cooperativity was lower (the values of Hill coefficients were 3.0 and 1.3 for Co2+ and Ni2+, respectively). Regulation of NADP-ME from tobacco leaves by divalent metal ions is discussed.
