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Recombinant DNA technology plays a vital role in improving health conditions by developing new pharmaceuticals.  Recently, some of the cytokines as other recombinant proteins could be produced using the recombinant DNA technology. The role of Recombinant IL-4 in allergy, autoimmunity, and cancer has been investigated. The present study was aimed to clone and produce the Human IL-4 under specific promoter in Escherichia coli and assessed its biological functions. The designed hIL-4 gene construct was artificially synthesized; subsequently, it was sub cloned into the pcDNA3.1 (+) vector in HindIII restriction enzyme site. Recombinant plasmid was transferred and expressed in BL21 cells. The rhIL-4 protein was evaluated by SDS-PAGE and Western blotting. It was purified by Ni-NTA affinity chromatography. The purified protein concentration and also accuracy were determined by ELISA. MTT assay was applied to evaluate the biological activity of rhIL-4 on the Erythroleukemic cell line proliferation. The rhIL-4 gene was successfully cloned and transformed into expression E. coli cells. As a result, a specific band was observed both on the SDS-PAGE and nitrocellulose membrane after Western blotting. The purified protein concentration was equal to 500 pg/ml. The MTT assay indicated that the exposed cells with rhIL-4 were proliferated in a dose dependent manner. The rhIL-4 gene under specific eukaryotic promoter was successfully cloned in the prokaryotic system and the transcription was carried out by T7 RNA polymerase. Therefore, mass production of IL-4 could be a great help in clinical trials and research studies. Additionally, prokaryotic system used in current work was less costly and less time-consuming. HIGHLIGHTS • Interleukin-4 (IL-4) is a cytokine that regulates multiple biological functions. • The recombinant Human IL-4 was cloned and produced under specific promoter in Escherichia coli. • Through MTT assay, the exposed cells with rhIL-4 were proliferated in a dose dependent manner.