Вариабельность накопления S-антигена вируса гепатита B в корнеплодах и листьях индивидуальных трансгенных растений моркови

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Вестник Томского государственного университета. Биология. 2016; : 126-139

Вариабельность накопления S-антигена вируса гепатита B в корнеплодах и листьях индивидуальных трансгенных растений моркови

Уварова Е. А., Загорская А. А., Поздняков С. Г., Дейнеко Е. В., Щелкунов С. Н.

https://doi.org/10.17223/19988591/33/8

Аннотация

Исследовано накопление поверхностного вирионного белка HBsAg вируса гепатита B человека в корнеплодах и листьях индивидуальных трансгенных растений моркови. В геном растений моркови был перенесен ген, кодирующий один из белков вирусной оболочки, а именно короткий S-антиген под управлением 35S промотора вируса мозаики цветной капусты Cauliflower mosaic virus (CaMV). Изучено соотношение количества HBsAg в листьях и корнеплодах у каждого индивидуального растения. Установлено, что среди изученных растений моркови только у двух соотношение между накоплением HBsAg в листьях и корнеплодах было близко к равнозначному. У других растений количество HBsAg в листьях преобладало над количеством в корнеплодах (восемь растений) или количество HBsAg в корнеплодах преобладало над количеством в листьях (три растения). Обсуждаются возможные причины отсутствия прямой корреляции между уровнем накопления HBsAg в тканях корнеплодов и листьев исследуемых трансгенных растений моркови.

Список литературы

1. Zuckerman J.N., Sabin C., Craig J., Williams A., Zuckerman A.J. Immune response to a new hepatitis B vaccine in healthcare workers who had not responded to standard vaccine: randomised double blind dose-response study // BMJ. 1997. Vol. 314, № 7077. P. 329-333.

2. Shouval D., Roggendorf H., Roggendorf M. Enhanced immune response to hepatitis B vaccination through immunization with a Pre-S1/Pre-S2/S Vaccine // Med. Microbiol. Immunol. 2015. Vol. 204, № 1. P. 57-68.

3. Brocke P., Schaefer S., Melber K., Jenzelewski V., Mutter F., Dahlems U., Bartelsen O., Park K.-N., Janowicz Z.A., Gellissen G. Recombinant Hepatitis B Vaccines: Disease Characterization and Vaccine Production // Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems / ed. Gellissen G. Wiley-VCH Verlag GmbH & Co. KGaA, 2004. P. 319-359.

4. Shouval D., Ilan Y., Adler R., Deepen R., Panet A., Even-Chen Z., Gorecki M., Gerlich W.H. Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-S1 and pre-S2 antigens as compared with conventional yeast-derived vaccines // Vaccine. 1994. Vol. 12, № 15. P. 1453-1459.

5. Mason H.S., Lam D.M., Arntzen C.J. Expression of hepatitis B surface antigen in transgenic plants // Proc. Natl. Acad. Sci. U. S. A. 1992. Vol. 89, № 24. P. 11745-11749.

6. Thanavala Y., Yang Y.F., Lyons P., Mason H.S., Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen // Proc. Natl. Acad. Sci. U. S. A. 1995. Vol. 92, April. P. 3358-3361.

7. Kapusta J., Modelska A., Figlerowicz M., Pniewski T., Letellier M., Lisowa O., Yusibov V., Koprowski H., Plucienniczak A., Legocki A.B. A plant-derived edible vaccine against hepatitis B virus // FASEB J. 1999. Vol. 13, № 13. P. 1796-1799.

8. Pniewski T. The twenty-year story of a plant-based vaccine against hepatitis B: Stagnation or promising prospects? // Int. J. Mol. Sci. 2013. Vol. 14, № 1. P. 1978-1998.

9. Sunil Kumar G.B., Ganapathi T.R., Revathi C.J., Prasad K.S.N., Bapat V.A. Expression of hepatitis B surface antigen in tobacco cell suspension cultures // Protein Expr. Purif. 2003. Vol. 32, № 1. P. 10-17.

10. Odell J.T., Nagy F., Chua N.-H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter // Nature. 1985. Vol. 313, № 6005. P. 810-812.

11. Battraw M.J., Hall T.C. Histochemical analysis of CaMV 35S promoter-P-glucuronidase gene expression in transgenic rice plants // Plant Mol. Biol. 1990. Vol. 15, № 4. P. 527-538.

12. Benfey P.N., Ren L., Chua N.H. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development // EMBO J. 1990. Vol. 9, № 6. P. 16771684.

13. Potenza C., Aleman L., Sengupta-Gopalan C. Targeting transgene expression in research, agricultural, and environmental applications: Promoters used in plant transformation // Vitr. Cell. Dev. Biol. - Plant. 2004. Vol. 40, № 1. P. 1-22.

14. Okumura A., Shimada A., Yamasaki S., Horino T., Iwata Y., Koizumi N., Nishihara M., Mishiba K. CaMV-35S promoter sequence-specific DNA methylation in lettuce // Plant Cell Rep. Springer Berlin Heidelberg, 2016. Vol. 35, № 1. P. 43-51.

15. Hansen G., Chilton M.D. Lessons in gene transfer to plants by a gifted microbe // Curr. Top. Microbiol. Immunol. 1999. Vol. 240. P. 21-57.

16. Mlynarova L., Keiser L.P.C., Stiekema W., Nap J.-P. Approaching the Lower Limits of Transgene Variability // Plant Cell. 1996. Vol. 8, № 9. P. 1589-1599.

17. Щелкунов С.Н., Саляев Р.К., Рыжова Т.С., Поздняков С.Г., Нестеров А.Е., Рекославская Н.И., Сумцова В.М., Пакова Н.В., Мишутина У.О., Копытина Т.В., Хэммонд Р.В. Создание кандидатной съедобной вакцины против вируса гепатита B и иммунодефицита человека на основе трансгенного томата // Вестник РАМН. 2004. № 11. С. 50-51.

18. Filipecki M., Malepszy S. Unintended consequences of plant transformation: a molecular insight. // J. Appl. Genet. 2006. Vol. 47, № 4. P. 277-286.

19. Дейнеко Е.В., Загорская А.А., Поздняков С.Г., Филипенко Е.А., Пермякова Н.В., Сидорчук Ю.В., Уварова Е.А., Позднякова Л.Д., Шумный В.К., Власов В.В., Хэммонд Р.В., Щелкунов С.Н. Анализ продукции М-антигена вируса гепатита B в листьях трансгенных растений моркови // Доклады РАН. 2009. Т. 425, № 3. С. 400403.

20. Schena M., Shalon D., DavisR.W., Brown P.O. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray // Science (80). 1999. Vol. 70, № 270. P. 467-470.

21. Galbraith D.W., Birnbaum K. Global studies of cell type-cpecific gene expression in plants // Annu. Rev. Plant Biol. 2006. Vol. 57, № 1. P. 451-475.

22. Brenner W.G., Schmulling T. Transcript profiling of cytokinin action in Arabidopsis roots and shoots discovers largely similar but also organ-specific responses // BMC Plant Biol. 2012. Vol. 12. P. 112.

23. Koncz C., Martini N., Szabadosz L., Hrouda M., Bachmair A., Schell J. Specialized vectors for gene tagging and expression studies // Plant Molecular Biolog. Manual / ed. by S.B. Gelwin, R.A. Schilperoort. Kluwer Academic Publishers, Dordrecht, Netherlands, 1994. P. 53-74.

24. Сидорчук Ю.В., Дейнеко Е.В., Власов В.В., Сенников С.В., Якушенко Е.В., Филипенко Е.А., Загорская А.А., Козлов В.А., Шумный В.К., Филипенко М.Л. Способ получения трансгенных растений моркови, продуцирующих интерлейкин-10 человека. Патент RU 2374321. 2009.

25. Murray M.G., Thompson W.F. Rapid isolation of high molecular weight plant DNA // Nucleic Acids Res. 1980. Vol. 8, № 19. P. 4321-4326.

26. Garbarino J.E., Belknap W.R. Expression of Its Promoter in Transgenic Plants // Plant Mol. Biol. 1994. Vol. 24. P. 119-127.

27. Malik K., Wu K., Li X-Q., Martin-Heller T., Hu M., Foster E., Tian L., Wang C., Ward K., Jordan M., Brown D., Gleddie S., Simmonds D., Zheng S., Simmonds J., Miki B. A constitutive gene expression system derived from the tCUP cryptic promoter elements // Theor. Appl. Genet. 2002. Vol. 105, № 4. P. 505-514.

28. Samac D.A., Tesfaye M., DornbuschM., SaruulP., Temple S.J. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa) // Transgenic Res. 2004. Vol. 13, № 4. P. 349-361.

29. Wally O., Jayaraj J., Punja Z.K. Comparative expression of P-glucuronidase with five different promoters in transgenic carrot (Daucus carota L.) root and leaf tissues // Plant Cell Rep. 2008. Vol. 27, № 2. P. 279-287.

30. Zheng X., Deng W., Luo K., Duan H., Chen Y., McAvoy R., Song S., Pei Y., Li Y. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters // Plant Cell Rep. 2007. Vol. 26, № 8. P. 1195-1203.

Tomsk State University Journal of Biology. 2016; : 126-139

Variability in the accumulation of hepatitis B virus S antigen in storage roots and leaves of individual transgenic carrot plants

Uvarova E. A., Zagorskaya A. A., Pozdnyakov S. G., Deineko E. V., Shchelkunov S. N.

https://doi.org/10.17223/19988591/33/8

Abstract

We transferred Gene env, encoding one of the proteins of the viral envelope, such as short S-antigen under control of the 35S promoter of cauliflower mosaic virus (CaMV), into the genome of carrot plants. The level of HBsAg in leaves and roots was examined as the ratio of HBsAg in the leaves and roots of thirteen carrot plants was determined by ELISA accumulation individually, and especially HBsAg accumulation in various plant organs. As a binary vector for plant transformation we used pBINPLUS/ARS plasmid (Fig. 1а), kindly provided by Dr. RW Hammond (USDA, USA). At sites of restriction endonucleases Hi«dIII-Acc65I we inserted HBsAg (GenBank V00867.1) coding sequence, under control of CaMV35S (435 bp) promoter and restricted at the 3'-end by the polyadenilation signal sequence мРНК CaMV (polyA, 202 bp), in pBINPLUS/ARS (Fig. 1b). Carrot cells (Daucus carota L.) were transformed by means of agrobacterium mediation. Genomic DNA of plants was isolated by the method of Murray MG and Thompson WF. The presence of the transformed gene in genomic DNA of carrot plants was confirmed by PCR using primer pairs (Fig. 1b) We observed variability both in leaves and roots of the transgenic carrot in HBsAg accumulation. The HBsAg content in the leaves of the plants chosen for this comparative analysis varied from 0.1 to 20.69 ng/g of fresh weight, amounting on the average to 9.43 ng/g, and in the storage roots, from 0.34 to 12.07 ng/g, with an average of 4.57 ng/g. HBsAg content in the plant leaves selected for this comparative analysis varied from 0.1 to 20.69 ng/g fresh weight, amounting, on average, to 9.43 ng/g, and in the storage roots, from 0.34 to 12.07 ng/g, with an average of 4.57 ng/g. Among analyzed plant carrots there are only two (№ 3 and № 25) where the relationship between HBsAg accumulation in leaves and roots was close to equal. Other plants were divided into two groups, the first group is where the ratio of HBsAg leaves prevailed over the number in the roots (eight plants) and the second group where the roots of HBsAg dominated the quantity in the leaves (three plants). We discussed possible reasons for the lack of a direct correlation between the level of HBsAg accumulating in the tissues of roots and leaves of the transgenic carrot. We found no direct correlation between the level of the target protein accumulation in leaves and storage roots of individual plants.

References

5. Mason H.S., Lam D.M., Arntzen C.J. Expression of hepatitis B surface antigen in transgenic plants // Proc. Natl. Acad. Sci. U. S. A. 1992. Vol. 89, № 24. P. 11745-11749.

8. Pniewski T. The twenty-year story of a plant-based vaccine against hepatitis B: Stagnation or promising prospects? // Int. J. Mol. Sci. 2013. Vol. 14, № 1. P. 1978-1998.

10. Odell J.T., Nagy F., Chua N.-H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter // Nature. 1985. Vol. 313, № 6005. P. 810-812.

11. Battraw M.J., Hall T.C. Histochemical analysis of CaMV 35S promoter-P-glucuronidase gene expression in transgenic rice plants // Plant Mol. Biol. 1990. Vol. 15, № 4. P. 527-538.

12. Benfey P.N., Ren L., Chua N.H. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development // EMBO J. 1990. Vol. 9, № 6. P. 16771684.

15. Hansen G., Chilton M.D. Lessons in gene transfer to plants by a gifted microbe // Curr. Top. Microbiol. Immunol. 1999. Vol. 240. P. 21-57.

16. Mlynarova L., Keiser L.P.C., Stiekema W., Nap J.-P. Approaching the Lower Limits of Transgene Variability // Plant Cell. 1996. Vol. 8, № 9. P. 1589-1599.

17. Shchelkunov S.N., Salyaev R.K., Ryzhova T.S., Pozdnyakov S.G., Nesterov A.E., Rekoslavskaya N.I., Sumtsova V.M., Pakova N.V., Mishutina U.O., Kopytina T.V., Khemmond R.V. Sozdanie kandidatnoi s\"edobnoi vaktsiny protiv virusa gepatita B i immunodefitsita cheloveka na osnove transgennogo tomata // Vestnik RAMN. 2004. № 11. S. 50-51.

18. Filipecki M., Malepszy S. Unintended consequences of plant transformation: a molecular insight. // J. Appl. Genet. 2006. Vol. 47, № 4. P. 277-286.

19. Deineko E.V., Zagorskaya A.A., Pozdnyakov S.G., Filipenko E.A., Permyakova N.V., Sidorchuk Yu.V., Uvarova E.A., Pozdnyakova L.D., Shumnyi V.K., Vlasov V.V., Khemmond R.V., Shchelkunov S.N. Analiz produktsii M-antigena virusa gepatita B v list'yakh transgennykh rastenii morkovi // Doklady RAN. 2009. T. 425, № 3. S. 400403.

20. Schena M., Shalon D., DavisR.W., Brown P.O. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray // Science (80). 1999. Vol. 70, № 270. P. 467-470.

21. Galbraith D.W., Birnbaum K. Global studies of cell type-cpecific gene expression in plants // Annu. Rev. Plant Biol. 2006. Vol. 57, № 1. P. 451-475.

24. Sidorchuk Yu.V., Deineko E.V., Vlasov V.V., Sennikov S.V., Yakushenko E.V., Filipenko E.A., Zagorskaya A.A., Kozlov V.A., Shumnyi V.K., Filipenko M.L. Sposob polucheniya transgennykh rastenii morkovi, produtsiruyushchikh interleikin-10 cheloveka. Patent RU 2374321. 2009.

25. Murray M.G., Thompson W.F. Rapid isolation of high molecular weight plant DNA // Nucleic Acids Res. 1980. Vol. 8, № 19. P. 4321-4326.

26. Garbarino J.E., Belknap W.R. Expression of Its Promoter in Transgenic Plants // Plant Mol. Biol. 1994. Vol. 24. P. 119-127.

Вариабельность накопления S-антигена вируса гепатита B в корнеплодах и листьях индивидуальных трансгенных растений моркови

Аннотация

Variability in the accumulation of hepatitis B virus S antigen in storage roots and leaves of individual transgenic carrot plants

Abstract

События