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ISSN 1027-2852 (Electronic)
ISSN 0864-4551 (Print)

2012, Number 2

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Biotecnol Apl 2012; 29 (2)

Generation of two polyclonal antibodies for western blot detection of hepatitis A virus in plants

Hernández A, Gonzalez A, López A, Rosabal Y, Ceballo Y, Soto J, Enríquez G

Language: English
References: 20
Page: 102-107
PDF size: 249.93 Kb.


ABSTRACT

In recombinant systems, the availability of good quality antibodies is required to detect Hepatitis A virus (HAV) proteins. Following this line, the cDNA sequences coding for two hepatitis A virus structural proteins, VP2 and VP3, were cloned into the bacterial expression vector pTrcHis yielding plasmids pTrcVP2 and pTrcVP3. Recombinant Escherichia coli XL1-blue strains harboring these two constructs were grown in liquid media and after IPTG gene induction, both fused recombinants molecules were detected by SDS-PAGE as 34 kDa and 24 kDa bands respectively. Recombinant VP2 and VP3 proteins containing N-terminal hexa-histidine tags were purified under denaturing conditions by immobilized metal affinity chromatography yielding 8 mg and 10 mg of refolded protein per 300 mL of bacterial culture respectively. Immunization of rabbits against purified recombinant proteins allowed the production of high titer polyclonal sera with immunological reactivity detecting hepatitis A virus proteins using western-blot analysis. According to our results, these two polyclonal sera constitute a valuable tool to follow the production of HAV proteins in transgenic plants, where a putative expression of HAV proteins higher than 0.25% of the total soluble protein could be detected with the use of these sera by western blot assay.


REFERENCES

  1. Gust ID, Coulepis AG, Feinstone SM, Locarnini SA, Moritsugu Y, Najera R, et al. Taxonomic classification of hepatitis A virus. Intervirology. 1983;20(1):1-7.

  2. Harmon SA, Updike W, Jia XY, Summers DF, Ehrenfeld E. Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli. J Virol. 1992;66(9):5242-7.

  3. Dzagurov GK, Kusov I, Gauss-Mueller V. Expression of hepatitis A virus procapsids in the insect cells infected by recombinant baculovirus. Vopr Virusol 2003;48(3): 36-40.

  4. Yao GFLC, Jiming RLZ. Expression of Hepatitis A virus proteins by recombinant vaccinia virus [J]. Chinese J Virol. 1989;4. Available from: http://en.cnki.com.cn/Article_en/CJFDTOTAL-BDXB198904000.htm

  5. López A, Rosabal Y, Hernández A, González B, Ríos J, Pérez M, et al. Expression of the Hepatitis A virus empty capsids in suspension cells and transgenic tobacco plants (Nicotiana tabacum L.). Biotecnol Apl 2008;25(1):42-6.

  6. National Research Council. Guide for the Care and Use of Laboratory Animals. 8th Edition. Washington, DC: The National Academies Press; 2011.

  7. Chung HY, Lee HH, Kim KI, Chung HY, Hwang-Bo J, Park JH, et al. Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice. Plant Cell Rep. 2011; 30(8):1513-21.

  8. Dawson GJ, Decker RH, Norton DK, Bryce WH, Whittington RO, Tribby, II, et al. Monoclonal antibodies to hepatitis A virus. J Med Virol 1984;14(1):1-8.

  9. Schofield DJ, Emerson SU, Purcell RH. Four chimpanzee monoclonal antibodies that neutralize hepatitis A virus. Drugs Fut 2003;28(2):137-42.

  10. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J. 2005;46(3):258-68.

  11. Lu L, Cheng A, Wang M, Jiang J, Zhu D, Jia R, et al. Polyclonal antibody against the DPV UL46M protein can be a diagnostic candidate. Virol J 2010;7:83.

  12. Grifantini R, Pagani M, Pierleoni A, Grandi A, Parri M, Campagnoli S, et al. A novel polyclonal antibody library for expression profiling of poorly characterized, membrane and secreted human proteins. J Proteomics. 2011;75(2):532-47.

  13. Burgess RR. Refolding solubilized inclusion body proteins. Methods Enzymol. 2009;463:259-82.

  14. Jang TH, Park HH. Generalized semi-refolding methods for purification of the functional death domain superfamily. J Biotechnol. 2011;151(4):335-42.

  15. Zhang Y, Ma Y, Yang M, Min S, Yao J, Zhu L. Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro. Protein Expr Purif. 2011;75(2):155-60.

  16. Johnston JM, Harmon SA, Binn LN, Richards OC, Ehrenfeld E, Summers DF. Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli. J Infect Dis. 1988; 157(6):1203-11.

  17. Gauss-Muller V, Zhou MQ, von der Helm K, Deinhardt F. Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response. J Med Virol. 1990; 31(4):277-83.

  18. Gauss-Muller V, Lottspeich F, Deinhardt F. Characterization of hepatitis A virus structural proteins. Virology 1986; 155(2):732-6.

  19. Ross BC, Anderson DA. Characterization of hepatitis A virus capsid proteins with antisera raised to recombinant antigens. J Virol Methods. 1991;32(2-3):213-20.

  20. Tiwari S, Verma PC, Singh PK, Tuli R. Plants as bioreactors for the production of vaccine antigens. Biotechnology Advances. 2009;27(4):449-67.




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