Cloning Of IL-10 Gene, Wean Immune Regulatory Cytokine, And Preparation Of Its Prokaryotic Recombinant Protein

OBJECTIVE Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses and effects of immune regulatory. Cloning hIL-10 and preparation of rhIL-10 protein will supply a stable basis for the researching on effects of IL-10 expression in pediatric inflammatory, allergic disease and related clinic application.METHODS With peripheral blood from a patient suffered from a acute allergy, IL-10 gene expression was in a high level in monocytes. PBMC were isolated to extract total RNA. Then, RT-PCR was carried out for IL-10 gene screening using total RNA as a template, specific primers which derived from GenBank data of hIL-10 as a primer for reverse transcription and PCR primers. Recombinant plasmid pcDNA/HisMax-IL10 was constructed, whom can be expressed in mammalian cells for experiments of gene transfer by non-viral vector.The recombinant prokaryotic expression vector, pTrcHis2B-hIL10 was subcloned by molecular hybridization. Induced by IPTG, hIL-10 gene was expressed in E. coli JM109. Prokaryotic recombinant proteins were extracted and purified by Ni-NTA resin which contained 6×His ligand. rhIL-10. SDS-PAGE was used for recombinant products analysis.RESULTS PCR products were verified by restrictive endonuclease digestion and DNA sequencing. No error existed in full-length ORF of hIL-10 screened by PCR. And the location of ML-10 ORF within MCS of vector was in a right direction. SDS-PAGE showed a satisfied amount and purification of recombinant protein.CONCLUSION Using one step RT-PCR, ML-10 gene can be conveniently amplified through total RNA as a template. rhIL-10 can be prepared quickly and effectively with prokaryotic expression vector pTrcHis2B-ML10. It supplied a basis for further research of a cytokine, ML-10.