Cloning and Identification of Full-length cDNA of Rat Interleukin-10 Gene

Background: Hepatic fibrosis runs a chronic course, so far, the effectiveness of medical therapy has not been satisfactory, and gene therapy has become the focus in this field. It has been reported that interleukin (IL)-10 has protective effect on liver and may prevent the development of fibrogenesis. Aims: To clone and identify the full-length cDNA of IL-10 gene in Sprague-Dawley (SD) rats, so as to pave the way for the construction of adenovirus recombinants carrying rat IL-10 gene and for the study of gene therapy on liver fibrosis. Methods: A pair of IL-10 DNA primers were designed and synthesized. The cDNA fragment encoding mature peptide of rat IL-10 was amplified from the SD rat splenic monocyte by reverse transcriptase polymerase chain reaction (RT-PCR). The production of amplification was ligated to pMD-18T vector and then transformed into the competent bacteria DH5α to construct the recombinant vector pMD-18T-IL-10. Results: Using the total RNA of SD rat splenic monocyte as template, the rat IL-10 cDNA fragment containing 540 bp was amplified by RT-PCR. Restriction endonuclease mapping using HindⅢ and KpnⅠshowed that the target gene was inserted into the recombinant plasmid, and the amplified fragment was concordant with the sequence of rat IL-10 gene through DNA sequencing, which suggested that there were no mutation occurred in the coding area. Conclusions: The full-length cDNA of rat IL-10 gene was successfully cloned and the recombinant vector pMD-18T-IL-10 was constructed.