人羧基肽酶H不同区段抗原的克隆表达及其初步应用

目的 构建人羧基肽酶H不同区段抗原的原核表达载体,诱导表达获得重组蛋白,初步验证人羧基肽酶H不同抗原区段在羧基肽酶H自身抗体检测中的应用价值。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,转化大肠埃希氏菌诱导表达重组蛋白,用重组蛋白作为包被抗原建立人羧基肽酶H自身抗体的间接ELISA方法,评价羧基肽酶H抗原在检测新诊断2型糖尿病患者抗羧基肽酶H自身抗体中的应用价值。结果 获得了羧基肽酶H三种不同区段抗原,其中42～476氨基酸区段为较理想的抗原。以该全长抗原作为包被抗原检测95例新诊断2型糖尿病患者,羧基肽酶H自身抗体阳性率为8.42%。结论 原核克隆表达的人羧基肽酶H 42～476氨基酸区段抗原具有良好的抗原性,可作为成人隐匿性自身免疫性糖尿病鉴别诊断的候选抗原。

Cloning,Expression and Diagnostic Application of Different Fragments of Human Carboxypeptidase

Objective To obtain different fragments of human carboxypeptidase H,and evaluate the diagnostic application of the recombination carboxypeptidase H in detecting autoantibody.Methods The coding gene of carboxypeptidase H was obtained by RT-PCR.The corresponding prokaryotic expression vectors were constructed and transformed into E.coli to induce the expression of the recombination different fragments of carboxypeptidase H.Using these antigen fragments as the coating antigens,the enzyme-linked immunosorbent assay(ELISA)was established for the detection of carboxypeptidase H autoantibody in 95 newly diagnosed type 2 diabetes patients.Results Three fragments of human carboxypeptidase H were obtained,in which the 42～476aa fragment antigen was ideal one.Using the full-length carboxypeptidase H as coating antigen,the positive rate of carboxypeptidaseH autoantibody was 8.42%.Conclusion Because of the favorable antigenicity,the 42～476aa fragment antigen of carboxypeptidase H couldbe the candidate antigen for discrimination and diagnosis of latent autoimmune diabetes in adults.