Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

Mina Mirian, Razieh Taghizadeh, Hossein Khanahmad, Mansour Salehi, Ali Jahanian-Najafabadi, Hojjat Sadeghi-aliabadi, Shirin Kouhpayeh

Abstract


Hepatitis B virus (HBV) is considered as a global health concern and hepatitis B surface antigen (HBsAg) is the most immunogenic protein of HBV. The purpose of this study was to evaluate the expression of HBsAg on the cell surface of human embryonic kidney cell line (HEK293T). After transformation of expression vector pcDNA/HBsAg to E.coli TOP10F’, plasmid was extracted and digested with BglII. Afterwards, the linearized vector was transfected to cells and treated with hygromycin B for 5 weeks to expand the resulted clonies. The permanent expression of HBsAg followed by flow cytometry uptill now about one year. Genomic DNA was extracted from transfected cells and the existence of HBsAg gene was assessed by PCR. Real-time RT-PCR was utilized to measure the expression at the RNA level and flow cytometery was carried out to assess protein expression. Insertion of HBsAg cDNA in HEK293T genome was confirmed by PCR. The results of real-time RT-PCR illustrated that each cell expresses 2275 copies of mRNA molecule. Flow cytometry showed that compared with negative control cells, 99.9% of transfected cells express HBsAg on their surface. In conclusion, stable expression of hepatitis B surface antigen on the membrane of HEK293T provides an accurate post-translational modification, proper structure, and native folding in contrast with purified protein from prokaryotic expression systems. Therefore, these exposing HBsAg cells are practical in therapeutic, pharmaceutical, and biological sets of research.


Keywords


Hepatitis B, HBsAg, Recombinant HEK293T cell

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In this research, a recombinant cell line (HEK293T/HBsAg) over-expressing HBsAg was constructed. The expression of HBsAg on the cell surface was characterized by flowcytometry. A highly and stably expressed HBsAg cell line could be applied in future animal immunization and cell SELEX projects to produce polyclonal and monoclonal antibodies, nanobodies and aptamer.

ACKNOWLEDGMENTS

The content of this paper is extracted from the Ph.D thesis NO. 394169 submitted by Mina Mirian which was financially supported by the Research Department of Isfahan University of Medical Sciences, Isfahan, I.R. Iran.

REFERENCES

Chevaliez S, Pawlotsky JM. Diagnosis and management of chronic viral hepatitis: antigens, antibodies and viral genomes. Best Pract Res Clin Gastroenterol. 2008;22(6):1031-1048.

Sitnik R, Pinho JRR, Bertolini DA, Bernardini AP, da Silva LC, Carrilho FJ. Hepatitis B virus genotypes and precore and core mutants in Brazilian patients. J Clin Microbiol. 2004;42(6):2455-2460.

World Health Organization (WHO). Hepatitis B, Fact sheet, updated July 2016. Available at: http://www.who.int/mediacentre/factsheets/fs204/en/ 2/8/2016.

Laub O, Rall L, Truett M, Shaul Y, Standring D, Valenzuela P, et al. Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region. J Virol. 1983;48(1):271-280.

Valenzuela P, Gray P, Quiroga M, Zaldivar J, Goodman HM, Rutter WJ. Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen. Nature. 1979;280(5725):815-819.

Even-Chen Z. Method for production and purification of hepatitis b vaccine. Google Patents. 1991.

Salisse J, Sureau C. A function essential to viral entry underlies the hepatitis B virus “a” determinant. J Virol. 2009;83(18):9321-9328.

Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat. 2004;11(2):97-107.

Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B: 2000--summary of a workshop. Gastroenterology. 2001;120(7):1828-1853.

McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature. 1984;307(5947):178-180.

Bailon P, Ehrlich GK, Fung WJ, Berthold W. Affinity Chromatography: Methods and Protocols. Humana Press. 2000.

Song KM, Lee S, Ban C. Aptamers and their biological applications. Sensors (Basel). 2012;12(1):612-631.

Vallian S, Khazaei MR. Medical applications of aptamers. Res Pharm Sci. 2007;2(2):59-66.

Mendonsa SD, Bowser MT. In vitro evolution of functional DNA using capillary electrophoresis. J Am Chem Soc. 2004;126(1):20-21.

Guo KT, Paul A, Schichor C, Ziemer G, Wendel HP. CELL-SELEX: Novel perspectives of aptamer-based therapeutics. Int J Mol Sci. 2008;9(4):668-678.

Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci U S A. 2006; 103(32):11838-11843.

Morris KN, Jensen KB, Julin CM, Weil M, Gold L. High affinity ligands from in vitro selection: complex targets. Proc Natl Acad Sci U S A. 1998;95(6):2902-2907.

Tuttleman JS, Pugh JC, Summers JW. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J Virol. 1986;58(1):17-25.

Elghanam MS, Attia AS, Shoeb HA, Hashem AE. Expression and purification of hepatitis B surface antigen S from Escherichia coli; a new simple method. BMC Res Notes. 2012;5:125.

Liu R, Lin Q, Sun Y, Lu X, Qiu Y, Li Y, et al. Expression, purification, and characterization of hepatitis B virus surface antigens (HBsAg) in yeast Pichia Pastoris. Appl Biochem Biotechnol. 2009;158(2):432-444.

Imamura T, Araki M, Miyanohara A, Nakao J, Yonemura H, Ohtomo N, et al. Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae. J Virol. 1987;61(11):3543-3549.

Lundstrom K, Wagner R, Reinhart C, Desmyter A, Cherouati N, Magnin T, et al. Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. J Struct Funct Genomics. 2006;7(2):77-91.

Gholson CF, Siddiqui A, Vierling JM. Cell surface expression of hepatitis B surface and core antigens in transfected rat fibroblast cell lines. Gastroenterology. 1990;98(4):968-975.

Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods. 2005;51(3):187-200.

De Groot AS, Scott DW. Immunogenicity of protein therapeutics. Trends Immunol. 2007;28(11):482-490.

Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5;172.

Khan KH. Gene expression in mammalian cells and its applications. Adv Pharm Bull. 2013;3(2):257-263.

Roossinck MJ, Jameel S, Loukin SH, Siddiqui A. Expression of hepatitis B viral core region in mammalian cells. Mol Cell Biol. 1986;6(5):1393-1400.

Lupberger J. Cultivation of Hepatitis B virus producing cell line HepG2.2.15 on microcarrier and functional characterization of the Hepatitis B virus polymerase. 2007.

Miller DG, Petek LM, Russell DW. Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet. 2004;36(7):767-773.


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