一种用于检测1型糖尿病的抗原蛋白质表达及鉴定方法的临床应用

Expression,identification and clinical application of an antigen protein for detection of type 1 diabetes mellitus

目的表达并鉴定一种用于检测1型糖尿病的抗原蛋白质,探讨其在化学发光检测系统中的临床应用。方法利用Primer 5.0软件设计引物,扩增谷氨酸脱羧酶65(GAD65)的部分蛋白质编码区(CDS)并克隆至pFastBac1载体中,经测序验证后在SF9细胞中表达。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定所得蛋白。利用该蛋白制备一种用于1型糖尿病检测的试剂盒,将其运用于化学发光检测系统,计算阳性检出率,并比较化学发光检测系统与蛋白质免疫印迹试验(Western blotting)阳性检出率的差异。结果成功表达了与GAD65抗体特异结合的抗原蛋白质,SDS-PAGE显示其相对分子质量为65000,与预测值相符;亲和层析纯化后蛋白纯度≥95%。所制备的化学发光检测试剂盒的阳性检出率明显高于Western blotting〔78.95%(30/38)比52.63%(20/38),P<0.05〕。结论GAD65抗体特异结合抗原蛋白纯度高、特异性强,利用该蛋白制备的相应试剂盒可应用于化学发光检测系统,提高了GAD65抗体的阳性检出率和对1型糖尿病的诊断效率,避免传统检测方法的漏检,值得在临床推广。

Objective To express and identify an antigen protein for the detection of type 1 diabetes mellitus,and investigate its application to the chemiluminescence detection system.Methods The partial coding sequence(CDS)region of glutamate decarboxylase 65(GAD65)was amplified by Primer 5.0 software and cloned into pFastBac1 vector.After sequencing,it was expressed in SF9 cells.The protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).A kind of kit for the detection of type 1 diabetes mellitus was prepared using this protein,it was applied to chemiluminescence detection system,the positive rate was calculated,and the positive rates of chemiluminescence immunoassay and Western blotting were compared.Results The specific binding protein of GAD65 antibody was successfully expressed.SDS-PAGE showed that its relative molecular mass was 65000,which was consistent with the predicted value,and the purity of protein was more than 95%.The positive rate of the prepared kit was significantly higher than that of Western blotting[78.95%(30/38)vs.52.63%(20/38),P<0.05].Conclusions The expressed protein of GAD65 antibody specific binding antigen has high purity and specificity.The corresponding kit can be used in the chemiluminescence detection system,and can improve the positive detection rate of GAD65 antibody,avoid the missed detection of traditional detection methods,and improve the diagnostic efficiency of type 1 diabetes,which is worthy of clinical promotion.