人白细胞介素10受体α基因真核表达及与JAK1蛋白的相互作用的检测

Eukaryotic Expression of Human Interleukin 10 Receptor α and Detection of Interactions in Protein JAK1s

构建含人白介素10受体α(IL-10RA)基因的真核表达质粒p ENTER-IL-10RA-His,并在HEK293中进行真核表达,并用免疫共沉淀检测JAK1与IL10RA在细胞内的相互作用。在He La细胞中提取人总RNA,通过RT-PCR获得人IL10RA的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR扩增、双酶切、测序鉴定后,将重组质粒p ENTER-IL-10RA-His转染至HEK293细胞中。免疫印迹法检测IL-10RA蛋白在HEK293细胞中的表达。结果显示,经PCR扩增和双酶切,测序鉴定质粒克隆正确。免疫印迹可见63 k D的目的蛋白。共同转染JAK1和IL-10RA的质粒,免疫印迹可见分别为133 k D和63 k D的目的条带,免疫共沉淀鉴定了JAK1和IL-10RA的相互作用。IL-10RA基因成功构建在p ENTER-His中,并在HEK293细胞中成功表达,并成功共转染JAK1和IL-10RA质粒,免疫共沉淀检测两者的相互作用。这为JAK1和IL-10RA相互作用的机制研究奠定基础。

This work is to construct a eukaryotic express vector pENTER-IL-IORA-His that expresses human interleukin 10 receptor α （ IL-10RA ） in HEK293 ceils, and then to detect the intracellular interaction between JAK1 and IL-10RA by co-immunoprecipitation. Human total RNA was extracted from Hela ceils, then IL-IORA gene was amplified by RT-PCR and inserted into eukaryotic vector pENTER-His. After validation by PCR, enzymatic digestion, and sequencing, the HEK293 ceils 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-IORA 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.