关键词: 白介素10受体α, （IL-10RA）, 真核表达, 免疫共沉淀, 蛋白相互作用

Abstract: This work is to construct a eukaryotic express vector pENTER-IL-10RA-His that expresses human interleukin 10 receptor α（IL-10RA）in HEK293 cells,and then to detect the intracellular interaction between JAK1 and IL-10RA by co-immunoprecipitation. Human total RNA was extracted from Hela cells,then IL-10RA gene was amplified by RT-PCR and inserted into eukaryotic vector pENTER-His. After validation by PCR,enzymatic digestion,and sequencing,the HEK293 cells were transfected with the recombined plasmid pENTER-IL-10RA-His. The expression level of IL-10RA at 48 h was determined by Western blotting. The results revealed that plasmid was cloned correctly,and the target protein of 63 kD was observed. When HEK293 cells were simultaneously transfected with the plasmid of both JAK1 and IL-10RA,the target protein band in 133 kD and 63 kD were identified. Co-immunoprecipitation validated the interaction between JAK1 and IL-10RA. In conclusion,IL-10RA gene was successfully constructed in recombined plasmid pENTER-His and expressed effectively in HEK293 cells. The interaction between JAK1 and IL-10RA was validated co-immunoprecipitation,which lays a foundation for further understanding the mechanism of interaction between JAK1 and IL-10RA.

Key words: interleukin 10 receptor α, （IL-10RA）,eukaryotic expression,co-immunoprecipitation,protein - protein interaction