Оцінка противірусної активності екстратів із трансгенних рослин тютюну з геном інтерферону a-2b людини

Abstract

Здійснено оцінку активності екстрактів із трансгенних рослин тютюну з геном інтерферону α-2b людини. Вміст інтерферону становив від 1017 до 2295 пг/г маси загального розчинного білка. При тестування екстрактів, отриманих з трансгенних рослин, на культурі клітин перещеплювальних текстикул поросят (ПТП), яка була інфіковані вірусом везикулярного стоматиту (ВВС), виявили інтерфероноподібну активність від 1,553×10 -3 до 3,009×10-3 МО/мг. Поряд із цим, рослини не набули стійкості до фітовірусних інфекцій. Інфікування трансгенних рослин різними фітовірусами призводило до розвитку захворювань.

Осуществлена оценка активности экстрактов из трансгенных растений табака с геном интерферона α-2b человека. Содержание интерферона составляло от 1017 до 2295 пг / г массы общего растворимого белка. При тестирование экстрактов, полученных из трансгенных растений, на культуре клеток перевивных текстикул поросят (ПТП), которые были инфицированы вирусом везикулярного стоматита (ВВС), обнаружили интерфероноподобную активность от 1,553 × 10 -3 до 3,009 × 10-3 МЕ / мг. Вместе с тем, растения не приобрели устойчивость к фитовирусных инфекций. Инфицирования трансгенных растений различными фитовирусами приводило к развитию заболеваний.

The presence of interferon in transgenic tobacco plants with human ifn-α2b gene and nptII was established using ELISA method. For the genetic transformation were used Nicotiana tabacum L.cv Petit Havana, grown with the Agrobacterium tumefaciens strain GV3101 PCV 124 vector with human gene ifn-α2b under the 35S promoter and the selective nptII gene. Tobacco explants were co-cultivated with a bacterial suspension on the MS medium with edition 2,5 mg / dm3 BAR; 0,05 mg / dm3 NAA, 500 mg / dm3 cefotaxime and 25 mg / dm3 kanamycin (selective medium) for plants regeneration. Received green shoots were cultivated on the medium half MS (MS where content of macrosols was changed in twice) with 25 mg / dm3 kanamitsin 500 mg / dm3 cefotaxime. Extracts obtained from dry material. The dry material was roasted with an aliquot of phosphate buffer (pH 7.4), which was centrifuged 15 min 15000 G for + 4C0. The supernatant was sampled to give a phosphate buffer (pH 7.4) in 1% dodecyl sulfate sodium and 1 mM PMSF, centrifuged at + 4С0 15min 15000 G. Determination of the antiviral activity of plant extracts was performed by inhibition of cytopathic action test virus in cell culture. As a test virus strain used VSV strain Indiana with infectious titer lgTTsD50 4.0 / 0.1 ml obtained from the Department of Virology museum Ministry of Health of Ukraine. As reference product used recombinant interferon ifn-α2b with antiviral activity of 100,000 IU / ml production NPO "Biopharma" Ukraine. The study was conducted micromethod in 96-well culture plates Sarstedt (Germany) Determination of interferon performed by the standard method using a set of regents ProCon100 produced by "Protein contour" (Russia). Alfalfa mosaic virus (AMV), tobacco mosaic virus (TMV), Potato virus Y (PVY), Potato virus X (PVX) and Potato virus A (PVA) mechanically inoculated into tobacco plants. After two weeks infection ELISA analysis was carried out for viruses (Gallo and Matisová, 1993). Primary antibodies (supplied by Dr. Cherovska, Prague) diluted 1: 1000 conjugated secondary alkaline phosphatase (anti-rabbit IgG) antibody (Sigma) diluted 1: 10,000 Imunoblotynh analyses were carried out using the same antibodies and dilutions. Number of interferon in plants was in range of 1017 to 2295 pg / g of total soluble protein. Interferon like activity of extracts from transgenic plants was showed in pigs cell culture, infected by vesicular stomatitis virus, and it was in range of 1,553 × 10 -3 to 3,009 × 10-3 IU / mg weight. However, after plants infection by fitoviruses, it was visually confirmed the symptoms develop of viral infection. Using ELISA and imunnoblot method, it has been noted the presence of target viral products in test plants. Despite, that all transgenic plants had an active interferon gene and their extracts have been showed an antiviral activity in cell culture, this plants were not resistant for plant virus interferon and also they could not stopped the infection development.