OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic c...OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.展开更多
胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是一种肠促胰岛素激素,具有很好的综合降糖效果,但其在血浆中的半衰期只有短暂几分钟,限制了GLP-1在临床方面的应用.为延长GLP-1的半衰期,合成了GLP-1-Fc融合基因,将N-糖基化作用的受...胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是一种肠促胰岛素激素,具有很好的综合降糖效果,但其在血浆中的半衰期只有短暂几分钟,限制了GLP-1在临床方面的应用.为延长GLP-1的半衰期,合成了GLP-1-Fc融合基因,将N-糖基化作用的受体天冬酰胺突变为丙氨酸,构建了无糖基化修饰的GLP-1-Fc(N126A)融合表达载体,利用酵母表达系统成功表达了GLP-1-Fc(N126A)融合蛋白,并利用r Protein A获得了高纯度融合蛋白.展开更多
基金supported by National Natural Science Foundation of China(81330081,81202596)Specialized Research Fund for the Doctoral Program of Higher Education(20123420110003)+1 种基金Program for Tackling Key Problems in Science and Technology by Anhui Province(1301042098)China Postdoctoral Science Foundation(2013M540508)
文摘OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.
基金This work is supported by the National Foundation forOutstanding Young Scientists(No.39825114) and the National Foun dation for"863"Project( 2001AA221021)
文摘胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是一种肠促胰岛素激素,具有很好的综合降糖效果,但其在血浆中的半衰期只有短暂几分钟,限制了GLP-1在临床方面的应用.为延长GLP-1的半衰期,合成了GLP-1-Fc融合基因,将N-糖基化作用的受体天冬酰胺突变为丙氨酸,构建了无糖基化修饰的GLP-1-Fc(N126A)融合表达载体,利用酵母表达系统成功表达了GLP-1-Fc(N126A)融合蛋白,并利用r Protein A获得了高纯度融合蛋白.
