人重组谷氨酸脱羧酶65基因的克隆表达及其在1型糖尿病诊断中的初步应用

目的 构建人重组谷氨酸脱羧酶65(GAD65)基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证GAD65不同抗原区段在1型糖尿病GAD自身抗体检测中的价值.方法 应用巢式逆转录聚合酶链式反应(RT-PCR)技术调取目的 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,用重组蛋白作为包被抗原,初步建立检测GAD自身抗体的酶联免疫吸附测定法(ELISA)方法,评价各片段在1型糖尿病诊断中的价值.结果 获得了4种可被1型糖尿病患者血清识别的重组人GAD抗原区段,其中GAD65(180-585)抗原区段具有很好的特异性,检出率为55.3%,是首选的抗原区段.结论 所选重组人GAD65(180-585)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.

Cloning,expression and diagnostic application for type 1 diabetes mellitus of human glutamic acid decarboxylase 65 gene

Objective To obtain different fragments of human glutamic acid decarboxylase 65 and to evaluate the diagnostic application for type 1 diabetes mellitus of the recombination human glutamic acid decarboxylase 65. Methods The coding gene of the glutamic acid decarboxylase 65 was obtained by RT-PCR. The corresponding prokaryotic expression vectors pBVILl/GAD65 were constructed and transformed into E.coli HBI01 to inducing the expression of the recombination human glutamic acid decarboxylase 65. Using these antigen fragments as the coating antigens, the enzyme-linked immunosorbent assay ( ELISA) was established for the detection of the glutamic acid decarboxylase autoantibody. Results The obtained four fragments of human glutamic acid decarboxylase 65 could react with the serum of type 1 diabetes mellitus patients. Because the specificity of the fragment of GAD65( 180-585) was the highest and the detection rate of this fragment was 55. 3% , the fragment of GAD65( 180-585) became the preferred antigen. Conclusion Because of the favourable antigenicity of the selected recombination human GAD65 (180-585), the GAD65 (180-585) could be the candidate antigen for developing the diagnostic reagent for type 1 diabetes mellitus.