CLONING AND EXPRESSION OF HUMAN IFNα2B GENE IN SACCHAROMYCES CEREVISIAE

摘要

Interferon is a protein secreted by eucaryotic cells following stimulation by viruses, bacteria, and many other immunogenes. Recent medical studies indicate that interferons have effective role in the treatment of virus infections, immunodeficiency and certain types of cancer such as hairy cell leukaemia (HCL). The aim of the present study is to apply yeast strain for secreting human IFNα2b following the use of yeast mating α factor signal peptide. On the other hand, cloning of IFNα2b gene without signal peptide and with non homologous signal peptide from Aspergillus was carried out as controls for comparison. First, human IFNα2b gene was amplified using mating α factor signal peptide codons and the first 20 bases of IFNα2b gene as a forward primer. This amplicon (700 bp) was cloned in pYZ4 vector and after using suitable restriction sites, the cleaved fragment was cloned in plasmid pYES2 as expression vector and named pPMSHβ2. Since the original construct of IFNα2b contained one of the Aspergillus signal peptides and flanked with Sal I and Eco RI sites, this gene (900 bp) was isolated and cloned in plasmid pET24d as an intermediate after using suitable sites. This insert was cleaved then cloned in pYES2 vector as pPMSHαB2. Also to construct IFNα2b without signal peptide, primers were designed to exclude the Aspergillus signal peptide from the original IFNα2b gene and the amplicon (500 bp) was cloned in plasmid pET23a then in pUC 18 and finally in plasmid pYES2 and named as pPMSH2. Saccharomyces cerevisiae (INVSC1) was transformed with these three mentioned constructs and for interferon gene expression, galactose was used as an inducer. Primary results of western blotting analysis showed that IFNα2b gene with α factor signal peptide was produced inside the pPMSHβ2 transformed yeast cells. The Use of α Factor signal peptide is convenient for expressing the IFNα2b gene in yeast. Studies on growth condition optimization and IFN secretion are under consideration and application.