Cloning of human interleukin-26 and constructing of its eukaryotic expression vector

Objective: To clone the gene of human interleukin-26(hIL-26)and to construct its eukaryotic expression vector.Methods: Total RNA was extracted from the human peripheral blood monocytes,and reverse transcriptase-polymerase chain reaction(RT-PCR)was performed.The PCR product was inserted into cloning vector pMD18-T to construct the recombinant plasmid of hIL-26——pMD18-T/hIL-26.Recombinants were transformed into E.coli-DH5α and screened with colony PCR and restriction analysis.The gene of hIL-26 cloned was sequenced.The hIL-26 gene fragment obtained from pMD18-T/hIL-26 was digested with SalⅠ and EcoRⅠ,and then inserted into pIRES2-EGFP that was cut with XhoⅠ and EcoRⅠ.Recombinants were identified by colony PCR and restriction analysis.The recombinant expression plasmid pIRES2-EGFP-hIL26 was transfected into COS7 cells mediated via nonliposomaltransfection reagent Fugene 6.Results: The sequence of the cloned DNA fragment was identical to hIL-26 that was reported on GenBank,and hIL-26 gene was inserted into eukaryotic expression vector pIRES2-EGFP correctly.The recombinant expression plasmid was successfully transfected into COS7 cells observed by fluorescent microscope and effective expression of hIL-26 was also testified by Western blotting.Conclusion: The recombinant eukaryotic expression vector of hIL26 is successfully constructed and effectively expressed in eukaryotic cells.