Cloning and expression of hepatitis B surface gene in E. coli

Krishma Gulzar, Maria Sharif, Arif M Khan, Muhammad Rizwan Javed, Imran Riaz Malik


Background: Hepatitis B virus (HBV) is among the smallest DNA viruses resulting in ~800,000 deaths each year. Pakistan is considered a country affected by HBV. In Pakistan, the most dominant genotype is D. HBV is an enveloped virus of 3.2 kb. The study's goal was to express hepatitis B surface antigen in a bacterial host to produce a recombinant protein.

Method: Blood samples were collected in EDTA coated vacutainer from patients after their consent. DNA was extracted from serum through the phenol-chloroform method; Hepatitis B surface gene was cloned in TA cloning vector, subclone in pET 28a expression vector. An expression vector containing the Surface gene was then transformed into a competent bacterial host BL21 and inducted with IPTG at 0.1-0.2mM concentration for expression. The expressed proteins (soluble and pellet form) were analyzed on SDS PAGE.

Results: Hepatitis B Surface gene of 681bp after PCR were detected under UV light then successfully cloned and subcloned in pET 28 expression vector. The restricted fragment indicating the gene of interest was 681bp when analyzed on 1.2% Agarose gel under UV light. The required protein of 25kDa was obtained in soluble form when detected on 12% SDS PAGE after staining with Coomassie Blue dye.

Conclusion: Hepatitis B surface gene was successfully expressed in both insoluble and pellet forms using E.coli. The expression of surface protein needs to maximize through optimizing conditions to be used as potent candidate for vaccine production to prevent hepatitis B infection.

Keywords: Hepatitis B virus; Surface gene; Cloning vector; pET expression vector   

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Tang CM, Yau TO, Yu J. Management of chronic hepatitis B infection: current treatment guidelines, challenges, and new developments. World Journal Gastroenterology, (2014); 20: 6262-6278.

Guidotti LG, Isogawa M, Chisari FV. Host-virus interactions in hepatitis B virus infections. Immunology, (2015); 36: 61-66.

Komatsu H. Hepatitis B virus: where do we stand and what is the next step for eradication? World Journal Gastroenterology, (2014); 20: 8998-9016.

Pourkarim MR, Olyaee SAB, Kurbanov F, Ranst MV, Tacke F. Molecular identification of hepatitis B virus genotypes/subgenotypes: Revised classification hurdles and updated resolutions. World Journal of Gastroenterology, (2014); 20 (23): 7152-7168.

Farhat M, Yasmeen A, Ahmad A. An overview of Hepatitis B and C in Pakistan. Journal of Microbiological Allied Sciences, (2014); 1(2): 98-102.

Majid M, Razza A, Anwar MA, Qayyum M, Zaman N, Khanum A, Beg MA. Analysis of Complete and Partial Genome Sequences of Hepatitis B Virus and Determination of its Genotypes and Sub-Genotypes from Pakistan. Pakistan Journal of Zoology, (2016); 48(3): 747-753.

Wei QK, Xiao T, Li J, Yin K, Jia F, Xu C, Zhao G, Cui Y, Liu G, Sun H, Jiang H, Yan G, Huang B. Construction and identification of Complex DNA vaccine of hepatitis B and Toxoplasma gondii. International Journal of Clinical and Experimental Medicine, (2015); 8(6): 9156-9161.

Huang CH, Chen E, Yang YY, Chen YY, Chang JJ, Chen KJ, Lu CH, Lee KD, Chen PC, Chen CC. The impact of hepatitis B virus infection and vaccination on the development of non-Hodgkin lymphoma. Journal of Viral Hepatitis, (2017); 10(24): 885-894.

Heryakusuma C, Puspasari F, Ihsanawati, Arifin E,Rachman G, Irasoniatan M, Ramadhani E, Nurainy N, Natalia D. Cloning and Expression of small Hepatitis B Surface Antigen (sHBsAg) in Hansenulla polymorpha. Microbiology Indonesia, (2016); 10: 119-124.

Wurm DJ, Veiter L, Ulonska S, Eggenreich B, Herwig C, Spadiut O. The E.coli pET expression system revisited mechanistic correlation between glucose and lactose uptake. Applied Microbiology and Biotechnology, (2016); 100(20): 8721-8729.

Malik A, Kumar D, Khan AA, Khan AA, Chaudhary AA, Husain SA, Kar P. Hepatitis B virus precore G1896A mutation in chronic liver disease patients with HBeAg negative serology from North India. Saudi journal of biological sciences, (2018); 25(7):1257-62.

Alhusseini NF, Abadeer MZ, El-Taher SM. Hepatitis B virus DNA can be amplified directly from blood spot on filter paper. American Journal of Biochemistry and Biotechnology, (2012); 2(8): 143-149.

Bandehpour M, Khodabandeh M, Kazemi B. Cloning and Expression of Hepatitis Surface Antigen. Hepatitis Monthly, (2008); 8(1): 17-21.

Zhang R, Xu Y, Xiao R, Wang S, Zhang B. Improved production of (R)-1-phenyl-1,2-ethanediol using Candida parapsilosis (R)-carbonyl reductase expressed in Pichia pastoris. Process Biochem. (2011); 46(3): 709-713.

Lui A, Gui S, Zhang L, Chen Z, Tang Y, Xiao M, Wang J, Liu W, Jin X, Zhu J, Lu X. Priduction of Bioactive-Liver targeting interferon Mu-IFN-CSP by soluble prokaryotic expression. AMB Express, (2017); 7: 192

Rizkia PR, Silaban S, Hasan K, Kamara DS, Subroto T, Soemitro S, Maksum IP. Effect of Isopropyl-β-D-thiogalactopyranoside concentration on prethrombin-2 recombinan gene expression in Escherichia coli ER2566. Procedia Chemistry, (2015); 17: 118-24.


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