Cloning and Heterologous Expression of Hepatitis B Virus Pre-Surface Antigen 1
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XPERIMENTAL WORKS
Cloning and Heterologous Expression of Hepatitis B Virus Pre-Surface Antigen 1 Rana Muhammad Mateena, *, Asma Tariqb, Muhammad Alia, Muhammad Sohail Afzala, Zahoor Qadir Samrab, and Muhammad Amin Atharb aDepartment
of Life Sciences, School of Science, University of Management and Technology, C-II Johar Town, Lahore, 54770 Pakistan b Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54000 Pakistan *e-mail: [email protected] Received September 9, 2019; revised September 24, 2019; accepted September 27, 2019
Abstract—Hepatitis B causes major deathly infections in developing countries. The surface antigen preS1, hepatocytes attachment site, plays a crucial role in development and progression of disease. The present study was, therefore, an effort to develop an efficient expression system for PreS1 gene in Esherishia coli. PreS1 gene was amplified and cloned in pTZ57R/T vector. Following confirmation through restriction enzyme digestion and sequencing, it was subcloned in pET22b(+). E. coli BL21 (DE3) CodonPlus cells were transformed with the recombinant plasmid. PreS1 gene was induced using IPTG, the protein was expressed and quantified with Bradford assay and analyzed on SDS-PAGE. Enzyme Linked Immunosorbent Assay and Western Blot analysis were performed for protein integrity and conformation. Keywords: ELISA, recombinant plasmid, SDS PAGE, western blot DOI: 10.3103/S089141682002007X
INTRODUCTION Hepatitis B is a life-threatening infection of liver which is caused by the hepatitis B virus. Hepatitis B virus (HBV) being one of the smallest viruses; presents a highly compact genetic organization [1]. Its DNA comprises four open-reading frames (ORF), which partially overlap, among which one ORF encodes the three envelope proteins. These hepatitis B surface antigens (HBsAg) are created from different initiation codons and are named as large, middle, and small protein represented as L, M and S, respectively. The M protein contains the S sequence with an extra N-terminal domain known as PreS2. The L protein being the largest one, contains S domain, PreS2 domain and an additional N-terminal domain PreS1 [2]. Recent studies have focused on PreS1 gene and its expression as it plays a central role in the life cycle of this virus. HBV attaches to the liver cell of the host through the contact of pre-S1 with the hepatocyte receptors [3]. It has been studied that pre-S sequence between Arg-103 and Ser-124 plays a central role in morphogenesis of HBV [4] and participates in virus assembly and excretion [5, 6]. Above all, it seems that the Pre-S deletions region are linked with the development of liver disease [7] causing decreased synthesis of minor surface antigens that are grouped in hepatocyte and endoplasmic reticulum (ER) leading to ER stress and hepatocellular carcinoma [8, 9]. Moreover, in
early infection, antibodies to pre-S1 were also detected, proving its strong immunogenicity. These antibodies pre-S1 region are represent an early stage biomarker for acute HBV inf
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