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目的构建人锌转运子ZnT8基因N末端、C末端和N-C融合区段的原核表达载体,诱导表达获得重组蛋白,并初步验证各抗原在1型糖尿病ZnT8自身抗体检测中的价值。方法应用RT-PCR方法调取目的基因,构建相应的原核表达质粒,转化大肠杆菌E.coli HB101,诱导表达获得纯化重组蛋白,并作为抗原包被酶联板,初步建立检测ZnT8自身抗体的ELISA方法 ,评价各基因片段在1型糖尿病诊断中的价值。结果获得了3种可被1型糖尿病患者血清识别的重组人ZnT8抗原区段,其中C末端区段ZnT8(268-369aa)与其它两个片段相比具有更高的检测敏感性和特异性,成为首选的抗原区段。结论所选重组人ZnT8(268-369aa)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原。
Objective To construct a prokaryotic expression vector for human zinc transporter ZnT8 gene at the N-terminus, C-terminus and N-C fusion region, and to obtain the recombinant protein by inducing the expression of ZnT8 autoantibodies against type 1 diabetes mellitus. Methods The target gene was picked up by RT-PCR, and the corresponding prokaryotic expression plasmid was constructed. The recombinant plasmid was transformed into E. coli HB101 and induced to express the recombinant protein. ELISA was used to coat the antigen. Methods to evaluate the value of each gene fragment in the diagnosis of type 1 diabetes mellitus. Results Three recombinant human ZnT8 antigen fragments were obtained, which were recognized by the serum of patients with type 1 diabetes. The C-terminal segment ZnT8 (268-369aa) had higher detection sensitivity and specificity than the other two fragments, Become the preferred antigen segment. Conclusion The selected recombinant human ZnT8 (268-369aa) antigen segment has good antigenicity and can be used as a candidate antigen for adjuvant diagnostic reagent in type 1 diabetic patients.