ISSN 2756-3413
African Journal of Virology Research ISSN 3421-7347 Vol. 11 (4), pp. 001-006, April, 2017. © International Scholars Journals
Full Length Research Paper
Subcloning and expression of human alpha-fetoprotein gene in Pichia pastoris
Mohammad Reza Mashayekhi 1,3, Nosratollah Zarghami1,3*, Delavar shahbazzadeh2, farzin Rohvand2, Mohammad Azizi2 and Behrang Alani1,3
1Department of Molecular Biochemistry and RIA, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2Department of Biotechnology, Pasteur Institute, Tehran, Iran.
3Tuberclosis and Lung Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran.
Accepted 11 November, 2016
Abstract
Alpha-fetoprotein protein (AFP) is naturally found in fetal serum while production and appearance after birth is indicative of presence of malignant tumors. Therefore, by measuring this protein during fetal period and after birth, it is possible to diagnose abnormalities and tumors in fetus or the newborn. The objective of this study was to produce and purify AFP protein using DNA recombinant technology to apply in diagnostic kit preparations. In this study Pichia pastoris as metylotrophic yeast was used for AFP production. After construction of recombinant plasmid, pS1-AFP, electroporation and lithium chloride techniques were used for transferring to susceptible cells. The quantity and quality of the produced protein were checked by SDS-PAGE and ELISA methods. Selection of transformed mutant strains of Muts and culturing them in glycerol media (YPG) up to OD600=6 and their transfer to methanol media (YPM) with augmentation of methanol to 1% final concentration resulted in inducing protein production in auxotrophic media lacking histidine. This protein could be useful in monoclonal antibody production and in diagnostic kit preparations.
Key words: Alpha-fetoprotein, Pichia pastoris, cloning, expression.