Design and Synthesis of Recombinant Murine Interleukin 4 (IL-4)

Background: Today’s use of the recombinant cytokines in the fields of diagnosis, treatment and research is greatly considered. With regards to the important role of the Interleukin-4 cytokine in modulating and assessment of immune response, the availability of recombinant mouse IL-4 (mIL-4) cytokines will provide an important tool for researchers to further studies. Method: Total RNA was extracted from mouse spleen tissue. cDNA was then synthesized using RT-PCR. The target sequence was amplified by PCR, using designed primers which contains restriction sites. Analysis of PCR product was done by agarose gel electrophoresis. Amplified sequence was ligated in to the pET21-b (+) vector. E. coli Top10 was transformed with recombinant plasmid pET21-b (+) vector.The cloning was verified by PCR and gene sequencing. BL21(DE3)-CodonPlus E. coli bacteria was transformed with recombinant plasmid pET21-b (+) vector for expression of protein. The expression of protein was induced by IPTG. The expressed protein was analysed with SDS-PAGE. After use of the guanidine hydrochloride and dithiothreitol, the protein was refolded and purified by chromatography. To demonstrate the nature of the protein, Interleukin-4, western blot analysis was performed. Results: The length of visualized PCR product band on electerophoresis was correspond to the target fragment (360 bp). Analysis of PCR product which have done for screening colonies showed 591 bp band that indicated the desired ligation and transformation of colonies. SDS-PAGE of supernatant and sediment from the lysis of bacteria showed the 17.5 kDa insoluble recombinant protein. SDS-PAGE analysis of the eluted fraction of chromatography showed that recombinant protein was completely purified. The binding of anti-Interleukin-4 specific antibody with recombinant protein was confirmed by immunoblotting. Conclusion: Recombinant mouse IL-4 precursor is expressed as an insoluble molecule with 17.5 kDa molecular weight by pET-21b (+) and BL21(DE3)-CodonPlus E. coli bacteria, which can be change to soluble form by denaturation with guanidine hydrochloride. Using Inoue method to competent bacteria have a higher efficiency than other methods. In addition, in this study the production of IL-4 at optimum temperature of 25 °C showed higher expression. Expressed recombinant protein after confirmation of it’s nature by western blotting and due to its high purity, could be used for diagnostic purposes.