Hernández A, López A, Ceballo Y, Rosabal L, Rosabal Y, Tiel K, Pérez M, González EM, Ramos O, Enríquez G

Language: English
References: 21
Page: 97-100
PDF size: 185.47 Kb.

ABSTRACT

The hepatitis B surface antigen (HBsAg) has been successfully produced in tobacco leaves, potato tubers and banana fruits, but at a rather low yield. Plant seeds are well suited for the accumulation, long-time storage and preservation of recombinant proteins with pharmaceutical interest, including antigens for prophylactic or therapeutic vaccines. In this work, we assessed the efficacy of the tissue-specific phaseolin promoter in driving the expression of the HBsAg gene in tobacco seeds. Two binary constructs were designed to synthesize this antigen fused or not to the KDEL motif for protein targeting to the endoplasmic reticulum. Tobacco plants were transformed via Agrobacterium tumefaciens using each construct. The highest yield (2.2 µg of the recombinant protein per gram of seed) was achieved by a transgenic line carrying the HBsAg gene without the KDEL fusion. The heterologous antigen aggregated properly forming particles that could be determined using an immunoenzymatic assay based on monoclonal antibody which recognizes the immunodominant epitope a of HBsAg, although seed-derived antigens sedimented slightly faster than the one produced in Pichia pastoris yeast. The HBsAg-expressing tobacco seeds could constitute a novel alternative source for the cost-effective production of the antigen.

REFERENCES

1. Kumar GB, Ganapathi TR, Bapat VA. Production of hepatitis B surface antigen in recombinant plant systems: an update. Biotechnol Prog. 2007;23(3):532-9.

2. Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA. 1992;89(24):11745-9.

3. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol. 2000;18(11):1167-71.

4. Kumar GB, Ganapathi TR, Revathi CJ, Srinivas L, Bapat VA. Expression of hepatitis B surface antigen in transgenic banana plants. Planta. 2005;222(3):484-93.

5. De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, et al. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat Biotechnol. 2002;20(12):1265-8.

6. Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, et al. Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci USA. 2007; 104(4): 1430-5.

7. Ramirez YJ, Tasciotti E, Gutierrez- Ortega A, Donayre Torres AJ, Olivera Flores MT, Giacca M, et al. Fruit-specifi c expression of the human immunodefi ciency virus type 1 tat gene in tomato plants and its immunogenic potential in mice. Clin Vaccine Immunol. 2007;14(6):685-92.

8. Horsch RB, Klee HJ. Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process. Proc Natl Acad Sci USA. 1986;83(12):4428-32.

9. Leyva A, Sánchez JC, López L, Font M, Gonzalez T, Pérez B, et al. Development, validation and application of a new ELISA for process control of the production of recombinant Hepatitis B surface antigen. Biotecnol Apl. 2011;28(4):228-34.

10. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5.

11. Eble BE, MacRae DR, Lingappa VR, Ganem D. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol Cell Biol. 1987;7(10):3591-601.

12. Shchelkunov SN, Shchelkunova GA. Plant-based vaccines against human hepatitis B virus. Expert Rev Vaccines. 2010;9(8):947-55.

13. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, et al. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010;8(5):588-606.

14. Kumar GB, Srinivas L, Ganapathi TR, Bapat VA. Hepatitis B surface antigen expression in transgenic tobacco (Nicotiana tabacum) plants using four different expression cassettes. Plant Cell Tiss Org Cult. 2006;84(3):315-23.

15. Morandini F, Avesani L, Bortesi L, Van Droogenbroeck B, De Wilde K, Arcalis E, et al. Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals. Plant Biotechnol J. 2011;9(8):911-21.

16. Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D, Higgins TJ. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J. 1992; 2(2):181-92.

17. Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Kunert R, Cao J, et al. Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Plant Biotechnol J. 2011;9(2):179-92.

18. Sojikul P, Buehner N, Mason HS. A plant signal peptide-hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells. Proc Natl Acad Sci USA. 2003;100(5):2209-14.

19. Huang Z, Mason HS. Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system. Plant Biotechnol J. 2004;2(3):241-9.

20. Felici F, Puchades Y, Fernández de Cossío ME, Vispo NS. Exposición multivalente utilizando la proteína pVIII de la cápsida de los fagos fi lamentosos. In: Nelson Santiago Vispo, editor. Biología combinatoria. La Habana: Elfos Scientiae; 2001. p. 69-89.

21. Thanavala Y, Yang YF, Lyons P, Mason HS, Arntzen C. Immunogenicity of transgenic plantderived hepatitis B surface antigen. Proc Natl Acad Sci USA. 1995;92(8):3358-61.