[Expression of hepatitis B virus core antigen gene region in yeast cell].
In this study, the core antigen (HBcAg) gene region of hepatitis B virus (HBV) was transformed and expressed into an eukaryotic expression vector by recombinant DNA technology in order to obtain the protein used in anti-HBc tests which is being one of the most important marker for the serodiagnosis of HBV infections. For this purpose, HBV-DNA positive patient sera were used as the source of viral nucleic acids, and the primers coding HBcAg gene region have been designed. After the amplification of HBcAg gene region by polymerase chain reaction (PCR), the amplicons purified by Invisorb Spin Rapid PCR Kit" (Invitek, Germany), were cloned to pYES2.1 plasmid via the TOPO TA expression kit (Invitrogen, USA) and this plasmid was transformed to competent bacteria (TOPO 10F' Escherichia coli) by CaCl2 method. After competent bacteria were grown on LB (Lysogeny Broth) agar media supplemented with ampicillin, the plasmid "pYES2.1 + HBcAg" were isolated and transformed to Saccaromyces cerevisiae via the "S.c. EasyComp Transformation Kit" (Invitrogen, USA). Finally, the expression of HBcAg by the yeast was confirmed with the use of in house ELISA method. Since the diagnostic kits used in our country for hepatitis B serology are usually imported products, this creates a great economical burden. Thus, the experience and knowledge that builds up following such studies will help to produce our own diagnostic products using our equity.
Yapar
Mehmet
M
Gülhane Askeri Tip Akademisi Tip Fakültesi, Tibbi Mikrobiyoloji Anabilim Dali, Viroloji Bilim Dali, Ankara. tabipks@yahoo.com
Sener
Kenan
K
Bedir
Orhan
O
Altayli
Ertan
E
Kubar
Ayhan
A
D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D006512:Hepatitis B Core Antigens / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D011933:Reagent Kits, Diagnostic / Q000191:economics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology; D014170:Transformation, Genetic; D014421:Turkey
D006512:Hepatitis B Core Antigens; D011933:Reagent Kits, Diagnostic