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目的构建人盘状结构域受体2(DDR2)与IgG Fc段嵌合受体的重组质粒(pFLAG-CMV2-Fc DDR2),检测其自主磷酸化水平。方法设计Fc DDR2克隆引物,以质粒pSec Tag2B-Fc DDR2为模板,PCR扩增IgG Fc段与DDR2跨膜区和胞内区的c DNA序列(Fc DDR2),将其插入pFLAG-CMV2真核表达载体中。PCR扩增鉴定并测序验证重组质粒pFLAG-CMV2-Fc DDR2后,将其转染人胚肾HEK293T细胞,Western blot法验证HEK293T细胞中目标蛋白Fc DDR2的表达效率。将pcDNA3.1-DDR2转染HEK293T细胞,给予2型胶原蛋白刺激后作为阳性对照,用小鼠来源抗DDR2抗体免疫沉淀`pFLAG-CMV2-Fc DDR2阳性的HEK293T细胞及胶原刺激的pcDNA3.1-DDR2阳性的HEK293T细胞,4G10抗体分析DDR2的磷酸化水平,评价Fc DDR2的自主磷酸化能力。结果重组质粒pFLAG-CMV2-Fc DDR2的PCR扩增产物为大小约2800 bp左右的片段,与预期相符,测序验证正确。Western blot结果提示pFLAG-CMV2-Fc DDR2转染后的HEK293T细胞,FLAG-Fc DDR2表达水平显著上调。免疫沉淀结合Western blot分析提示FcDDR2的磷酸化水平与胶原刺激后的DDR2水平相当。结论成功构建了pFLAG-CMV2-FcDDR2真核表达载体,Fc DDR2的自主磷酸化与胶原蛋白刺激后DDR2的活化水平相当,可以用作DDR2经胶原蛋白刺激后的活化替代形式。
Objective To construct a recombinant plasmid (pFLAG-CMV2-Fc DDR2) containing the chimeric receptor of human discoid domain receptor 2 (DDR2) and IgG Fc fragment and detect its autophosphorylation. Methods Fc DDR2 cloning primer was designed. The Fc region of IgG Fc fragment, DDR2 transmembrane region and intracellular region were amplified by PCR using plasmid pSec Tag2B-Fc DDR2 as a template, and then inserted into pFLAG-CMV2 eukaryotic expression vector Carrier. The recombinant plasmid pFLAG-CMV2-Fc DDR2 was identified by PCR amplification and sequenced, then transfected into human embryonic kidney HEK293T cells. Western blot was used to verify the expression efficiency of Fc DDR2 in HEK293T cells. HEK293T cells were transfected with pcDNA3.1-DDR2 and stimulated with type 2 collagen as a positive control. The pFLAG-CMV2-Fc DDR2-positive HEK293T cells were immunoprecipitated with mouse-derived anti-DDR2 antibody and collagen-stimulated pcDNA3.1- DDR2-positive HEK293T cells, the 4G10 antibody was analyzed for the phosphorylation level of DDR2 to evaluate the autophosphorylation capacity of Fc DDR2. Results The PCR product of recombinant plasmid pFLAG-CMV2-Fc DDR2 was approximately 2800 bp in size and was in line with expectation. The sequencing was verified correctly. Western blot results showed that FLAG-Fc DDR2 expression was significantly up-regulated in HEK293T cells transfected with pFLAG-CMV2-Fc DDR2. Immunoprecipitation combined with Western blot analysis indicated that the level of FcDDR2 phosphorylation was comparable to the level of DDR2 after collagen stimulation. Conclusion The pFLAG-CMV2-FcDDR2 eukaryotic expression vector was successfully constructed. The autophosphorylation of Fc DDR2 was comparable to the activation of DDR2 after collagen stimulation, and could be used as an alternative to DDR2 activation after collagen stimulation.