摘要
目的运用基因工程技术获取具有免疫原性的谷氨酸脱羧酶（ＧＡＤ６５）重组蛋白，探讨其用于Ⅰ型糖尿病早期诊断的可行性。方法通过ＰＣＲ扩增获得ＧＡＤ６５全编码基因，经克隆及序列分析后在原核细胞中表达，鉴定表达产物的免疫原性，用表达产物初步检测３０例糖尿病病人血清中抗ＧＡＤ６５抗体。结果所获ＰＣＲ产物经序列分析确证为１７５８ｂｐ，编码５８５个氨基酸，并正确地重组到ｐＧＥＸ３Ｘ表达型质粒中，其在原核细胞中表达的融合蛋白具有免疫原性，并能应用于糖尿病病人血清中抗ＧＡＤ６５抗体的检测。结论利用具有免疫原性的ＧＡＤ６５基因重组蛋白，通过间接ＥＬＩＳＡ检测糖尿病病人血清中的相关抗体，是一种简便可行的Ⅰ型糖尿病早期诊断的方法。
Objective To get recombinant GAD 65 proteins which have immunogenicity by the method of gene engineering so that the recombinant protein can be used for early diagnosis in type I diabetes mellitus. Method We amplified the complete fragment encoding GAD65 gene by PCR, and expressed its protein in E.coli. DH5 α after inserting it in the vector and determining its nucleotide sequence. Consequentially, we attempted to prove the immunogenicity of expresion products and construct a method for detecting antibodies against GAD65 in Diabetic serum. Result The sequence analysis showed that the amplified fragments contained 1758bp, encoded 585 amino acid, and had been correctly inserted into pGEX 3X vector. The recombinant proteins expressed in E. Coli. DH5 α had immunogenicity and could be used to detect agtibodies against GAD65 in diabetic serum. Conclusion We have got recombinant GAD65 proteins which have immunogenicity and have used them to detect preliminarily antibodies against GAD65 in diabetic serum.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1998年第4期272-274,共3页
National Medical Journal of China
基金
卫生部科研基金
