ساب كلونينگ و بيان ژن آلفافيتو پروتيين انساني در مخمر پيكيا پاستوريس

Subcloning and Expression of Human Alphafetoprotein Gene in Pichia Pastotris Yeast

Background and Objectives: Alpha-fetoprotein protein is naturally found in fetal serum while production and appearance of that after birth is indicative of presence of malignant tumors. Therefore, with measurement of this protein during fetal period and after birth, it is possible to diagnose fetal abnormalities and tumors. Therefore the objective of this study was to produce and purify AFP protein using DNA recombinant technology for applying in diagnostic kits. Materials and methods: In this study Pichia pastoris as metylotrophic yeast were used for producing AFP protein. For amplifying of interest gene PUC18 vector and for protein expression pHIL-S l vector were used. After creating recombinant plasmid of pS 1 -AFP electroporation and lithium chloride methods were used for transferring to susceptible strains of GS 115-HIS Exotrophic media lacks of histidine were used for screening. In order to identify the cloned gene to yeast genome and produced phenotypes PCR method was applied. The two used culture media were YPG and YPM. The quantity and quality of produce protein were checked by SDS-PAGE and ELISA methods. Results: Restriction analysis of pS1-AFP recombinant and transformed plasmid revealed that contain a piece of 1.78 kbp AFP gene and was shown on 1% agarose gel electrophoresis. That was also confirmed by PCR method. Selection of transformed mutant strains of mut^3 in comparison to mut + and culturing of those in glycerol media (YPG) until OD 0=6 and then transferring to methanol media (YPM) with addition methanol of 1% final concentration resulted in inducing protein production in exotrophic media with lack of histidine. The rate of induced protein was 10 miug/mL. Conclusion: This study was shown that including acid phosphatase gene as a signal and promoter AOXI at the beginning of AFP gene was suitable for expression and secretion. Therefore, it is postulated that it could be useful for monoclonal antibody producing and designing diagnostic kit.