Expression and detection of the FMDV VP1 transgene and expressed structural protein in Arabidopsis thaliana
To explore the feasibility of developing a new type of plantderived foot-and-mouth disease virus (FMDV) oral vaccine, the plant seed-specific expression vector p7SBin438/VP1 carrying the VP1 gene of the FMDV strain O/China/99 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. This strain was used for transformation of Arabidopsis thaliana via the floral-dip method. The kanamycin-resistant transgenic plants were selected, and the VP1 gene and protein expressions were confirmed by PCR and sandwich-ELISA, respectively. The ELISA positive plants were further confirmed by Western blot assay, and the VP1 gene in the transgenic progeny was confirmed by PCR. The results showed that the Agrobacterium-mediated floral-dip transformation method could produce transgenic Arabidopsis containing the VP1 gene. Therefore, this protocol provides a successful strategy to deliver viral genes to leguminous plants for genetic studies and for production of low-cost oral vaccines.
Expression and detection of the FMDV VP1 transgene and expressed structural protein in Arabidopsis thaliana
To explore the feasibility of developing a new type of plantderived foot-and-mouth disease virus (FMDV) oral vaccine, the plant seed-specific expression vector p7SBin438/VP1 carrying the VP1 gene of the FMDV strain O/China/99 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. This strain was used for transformation of Arabidopsis thaliana via the floral-dip method. The kanamycin-resistant transgenic plants were selected, and the VP1 gene and protein expressions were confirmed by PCR and sandwich-ELISA, respectively. The ELISA positive plants were further confirmed by Western blot assay, and the VP1 gene in the transgenic progeny was confirmed by PCR. The results showed that the Agrobacterium-mediated floral-dip transformation method could produce transgenic Arabidopsis containing the VP1 gene. Therefore, this protocol provides a successful strategy to deliver viral genes to leguminous plants for genetic studies and for production of low-cost oral vaccines.
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- Snowdon, W.A.: Th e susceptibility of some Australian fauna to infection with foot-and-mouth disease virus. Aust. J. Exp. Biol. Med. Sci., 1968; 46: 667-87.
- Aggarwal, N., Zhang, Z., Cox, S., Statham, R., Alexandersen, S., Kitching, R.P.: Experimental studies with foot-and-mouth disease virus, strain O, responsible for the 2001 epidemic in the United Kingdom. Vaccine, 2002; 20: 2508-15.
- Beck, E., Strohmaier, K.: Subtyping of European foot- and-mouth disease virus strains by nucleotide sequence determination. J. Virol., 1987; 61: 1621-9.
- Chan, E.W.C., Wong, H.T., Cheng, S.C.S., Yan, W.Y., Zhang, Z.X., Sheng, Z.T., Zhu, L.Q., Xie, Y.: An immunoglobulin G based chimeric protein induced foot-and-mouth disease specifi c immune response in swine. Vaccine, 2000; 19: 538-46. Lauterslager, T.G., Hilgers, L.A.: Effi cacy of oral administration and oral intake of edible vaccines. Immunol. Lett., 2002; 84: 185-90.
- Korban, S.S., Krasnyanski, S.F., Buetow, D.E.: Foods as production and delivery vehicles for human vaccines. Am. Coll. Nutr., 2002; 21: 212-7.
- Carrillo, C., Wigdorovitz, A., Trono, K., Dus Santos, M.J., Castanon, S., Sadir, A. M., Ordas, R., Escribano, J.M., Borca, M.V.: Induction of a virus-specifi c antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants. Viral. Immunol., 2001; 14: 49-57.
- Dus Santos, M.J., Wigdorovitz, A., Trono, K., Rios, R.D., Franzone, P.M., Gil, F., Moreno, J., Carrillo, C., Escribano, J.M., Borca, M.V.: A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine, 2002; 20: 1141-7.
- Wigdorovitz, A., Carrillo, C., Dus Santos, M.J., Trono, K., Peralta, A., Gomez, M. C., Rios, R.D., Franzone, P.M., Sadir, A.M., Escribano, J.M., Borca, M.V.: Induction of a protective antibody to foot and mouth disease virus in mice following oral of parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology, 1999; 236: 347-53.
- Sun, M., Qian, K.X., Su, N., Chang, H.Y., Liu, J.X., Chen, G.F.: Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol. Lett., 2003; 25: 1087-92.
- Pan, L., Zhang, Y.G., Wang, Y.L., Wang, B.Q., Wang, W.X., Fang, Y.Z., Jiang, S. T., Lv, J.L., Wang, W., Sun, Y., Xie, Q.G.: Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot- and-mouth disease virus elicit a protective response in guinea pigs. Immunol. Immunop., 2008; 121: 83-90.
- Mason, H.S., Ball, J.M., Shi, J.J., Jiang, X., Estes, M.K., Arntzen, C.J.: Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity. Proc. Natl. Acad. Sci. USA., 1996; 93: 5335-40.
- Van, R., Moloney, G.J.H.: Plant seed oil-bodies as carries for foreign proteins. Bio. Technology., 1995; 13: 72-7.
- Nain, V., Verma, A., Kumar, N., Sharma, P., Ramesh, B., Kumar, P.A.: Cloning of an ovule specifi c promoter from Arabidopsis thaliana and expression of beta-glucuronidase. Indian. J. Exp. Biol., 2008; 46: 207-11.
- Wang, G.L., Fang, H.J.: Plant Biotechnology, second ed. Science Press, Beijing, 2002; pp: 360-401.
- Ye, G.N., Deborah, S., Pang, S.Z.: Arabidopsis ovule is the target for Agrobacterium in planta vacuum infi ltration transformation. Plant. J., 1999; 19: 249-67.
- Clough, S.J., Bent, A.F.: Floral dip: a simplifi ed method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant. J., 1998; 16: 735-43.
- Xie, Q.G., McCahon, D., Crowther, J.R., Belsham, G.J., McCullough, K.C.: Neutralization of foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites. J. Gen. Virol., 1987; 68: 1637-47.
- Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W., Chua, N.H.: Agrobacterium-mediated transformation of Arabidopis thaliana using the fl oral dip method. Nat. Protoc., 2006; 1: 641- 6.
- Zale, J.M., Agarwal, S., Loar, S., Steber, C.M.: Evidence for stable transformation of wheat by fl oral dip in Agrobacterium tumefaciens. Plant. Cell. Rep., 2009; 28: 903-13.
- Richter, L.J., Th anavala, Y., Arntzen, C.J., Mason, H.S.: Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol., 2000; 18: 1167-71.
- Ruf, S., Hemann, M., Berger, I.J., Carrer, H., Bock R.: Stable genetic transformation of tomato plastids and expression of foreign protein in fruit. Nat. Biotechnol., 2001; 19: 870-5.
- Chen, Z.L.: Plant Biotechnology. Beijing University Press, Beijing, 1993; pp: 245-308.
- Shirasawa-Seo, N., Sano, Y., Nakamura, S., Murakami, T., Seo, S., Ohashi, Y., Hashimoto, Y., Matsumoto, T.: Characteristics of the promoters derived from the single-stranded DNA components of Milk vetch dwarf virus in transgenic tobacco. J. Gen. Virol., 2005; 86: 1851-60.
- Rosales-Mendoza, S., Alpuche-Solis, A.G., Soria-Guerra, R.E., Moreno-Fierros, L., Martinez-Gonzalez, L., Herrera-Diaz, A., Korban, S.S.: Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants. Plant. J., 2009; 57: 45-54.
- Kang, T.J., Han, S.C., Jang, M.O., Kang, K.H., Jang, Y.S., Yang, M.S.: Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Appl. Biochem. Biotechnol., 2004; 117: 175-87.
- Hohnjec, N., Lenz, F., Fehlberg, V., Vieweg, M.F., Baier, M.C., Hause, B., Küster, H.: Th e signal peptide of the Medicago truncatula modular nodulin MtNOD25 operates as an address label for the specifi c targeting of proteins to nitrogen-fi xing symbiosomes. Mol. Plant. Microbe. Interact., 2009; 22: 63-72.
- Wang, H.H., Yin, W.B., Hu, Z.M.: Advances in chloroplast engineering. J. Genet. Genomics, 2009; 36: 387-98.
- Kim, E.H., Suh, S.C., Park, B.S., Shin, K.S., Kweon, S.J., Han, E.J., Park, S.H., Kim, Y.S., Kim, J.K.: Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection. Planta., 2009;