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
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摘要
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 p ENTER-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-10 RA by co-immunoprecipitation. Humantotal RNA was extracted from Hela cells,then IL-10 RA gene was amplified by RT-PCR and inserted into eukaryotic vector p ENTER-His. Aftervalidation by PCR,enzymatic digestion,and sequencing,the HEK293 cells were transfected with the recombined plasmid p ENTER-IL-10RA-His. The expression level of IL-10 RA at 48 h was determined by Western blotting. The results revealed that plasmid was cloned correctly,and the target protein of 63 k D was observed. When HEK293 cells were simultaneously transfected with the plasmid of both JAK1 and IL-10 RA,the target protein band in 133 k D and 63 k D were identified. Co-immunoprecipitation validated the interaction between JAK1 and IL-10 RA. In conclusion,IL-10 RA gene was successfully constructed in recombined plasmid p ENTER-His and expressed effectively in HEK293 cells. The interaction between JAK1 and IL-10 RA was validated co-immunoprecipitation,which lays a foundation for further understanding themechanism of interaction between JAK1 and IL-10 RA.
This work is to construct a eukaryotic express vector p ENTER-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-10 RA by co-immunoprecipitation. Humantotal RNA was extracted from Hela cells,then IL-10 RA gene was amplified by RT-PCR and inserted into eukaryotic vector p ENTER-His. Aftervalidation by PCR,enzymatic digestion,and sequencing,the HEK293 cells were transfected with the recombined plasmid p ENTER-IL-10RA-His. The expression level of IL-10 RA at 48 h was determined by Western blotting. The results revealed that plasmid was cloned correctly,and the target protein of 63 k D was observed. When HEK293 cells were simultaneously transfected with the plasmid of both JAK1 and IL-10 RA,the target protein band in 133 k D and 63 k D were identified. Co-immunoprecipitation validated the interaction between JAK1 and IL-10 RA. In conclusion,IL-10 RA gene was successfully constructed in recombined plasmid p ENTER-His and expressed effectively in HEK293 cells. The interaction between JAK1 and IL-10 RA was validated co-immunoprecipitation,which lays a foundation for further understanding themechanism of interaction between JAK1 and IL-10 RA.
引文
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[2]von Lanzenauer SH,Wolk K,Kunz S,et al.Interleukin-10receptor-1 expression in monocyte-derived antigen-presentingcell populations:dendritic cells partially escape from IL-10’sinhibitory mechanisms[J].Genes&Immunity,2015,16(1):8-14.
[3]Trifunovi?J,Miller L,Debeljak Z,et al.Pathologic patterns ofinterleukin 10 expression--a review[J].Biochemia Medica,2015,25(1):36-48.
[4]Kim R,Emi M,Tanabe K,et al.Tumor-driven evolution ofimmunosuppressive networks during malignant progression[J].Cancer Research,2006,66(11):5527-5536.
[5]Darnell JJ,Kerr IM,Stark GR.Jak-STAT pathways andtranscriptional activation in response to IFNs and other extracellularsignaling proteins[J].Science,1994,264(5164):1415-1421.
[6]Beser OF,Conde CD,Serwas NK,et al.Clinical features ofinterleukin 10 receptor gene mutations in children with very earlyonset inflammatory bowel disease[J].Journal of PediatricGastroenterology&Nutrition,2014,60(3):332-338.
[7]Pestka S,Krause CD,Sarkar D,et al.Interleukin-10 and relatedcytokines and receptors[J].Annual Review of Immunology,2004,22:929-979.
[8]Commins S,Steinke JW,Borish L.The extended IL-10 superfamily:IL-10,IL-19,IL-20,IL-22,IL-24,IL-26,IL-28,and IL-29[J].Journal of Allergy&Clinical Immunology,2008,121(5):1108-1111.
[9]Stelloo E,Versluis MA,Nijman HW,et al.Microsatellite instabilityderived JAK1 frameshift mutations are associated with tumor immuneevasion in endometrioid endometrial cancer[J].Oncotarget,2016,7(26):39885-39893.
[10]Song B,Zhan H,Bian Q,et al.Piperlongumine inhibits gastriccancer cells via suppression of the JAK1,2/STAT3 signalingpathway[J].Molecular Medicine Reports,2016,13(5):4475-4480.
[11]Li Q,Gu S,Li N,et al.Protein interaction network construction andbiological pathway analysis related to atherosclerosis][J].ShengWu Yi Xue Gong Cheng Xue Za Zhi,2015,32(6):1255-1260.
[12]Verhelst J,De Vlieger D,Saelens X.Co-immunoprecipitation of theMouse Mx1 Protein with the Influenza A Virus Nucleoprotein[J].J Vis Exp,2015(98):e52871.
[13]Duan JW,Lu Z,Jiang B,et al.Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase(JAK)inhibitors[J].Bioorganic&Medicinal Chemistry Letters,2014,24(24):5721-5726.
[14]Einarson MB,Pugacheva EN,Orlinick JR.GST Pull-down[J].CSH Protoc,2007(2007):t4757.
[15]Valero ML,Sendra R,Pamblanco M.Tandem affinity purificationof histones,coupled to mass spectrometry,identifies associatedproteins and new sites of post-translational modification inSaccharomyces cerevisiae[J].J Proteomics,2016,136:183-192.
[16]Saito Y,Nakagawa T,Kakihana A,et al.Yeast Two-Hybridand One-Hybrid screenings identify regulators of hsp70 geneexpression[J].J Cell Biochem,2016,117(9):2109-2117.
[17]Yu Y,Li Y,Zhang Y.Yeast Two-Hybrid screening for proteinsthat interact with the extracellular domain of amyloid precursorprotein[J].Neurosci Bull,2016,32(2):171-176.
[18]Zhu Y,Chen X,Liu Z,et al.Interleukin-10 protection againstlipopolysaccharide-induced neuro-inflammation and neurotoxicityin ventral mesencephalic cultures[J].International Journal ofMolecular Sciences,2016,17(1).
[19]Wagner AH,Conzelmann M,Fitzer F,et al.JAK1/STAT3 activationdirectly inhibits IL-12 production in dendritic cells by preventingCDK9/P-TEFb recruitment to the p35 promoter[J].BiochemPharmacol,2015,96(1):52-64.
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