谷氨酸脱羧酶基因的体外表达

The expression of glutamic acid decarboxylase gene in vitro

目的 为获得足量的、可供实验研究和临床应用的谷氨酸脱羧酶（ Ｇ Ａ Ｄ６５） 抗原。方法将 Ｇ Ａ Ｄ６５ 基因的ｃ Ｄ Ｎ Ａ 正确地重组到表达型载体ｐ Ｇ Ｅ Ｘ３ Ｘ 中，并通过原核细胞进行诱导表达， Ｓ Ｄ Ｓ聚丙烯酰胺凝胶电泳测定表达产物分子量，间接 Ｅ Ｌ Ｉ Ｓ Ａ 法证实其免疫原性。结果 表达产物分子量约为６３ ．２３ Ｋ Ｄ，在误差允许范围（ ＜ ８ ％ ） 内，与 Ｇ Ａ Ｄ６５ 蛋白分子量相近，间接 Ｅ Ｌ Ｉ Ｓ Ａ 显示表达产物与１ 型糖尿病病人血清发生明显的显色反应，证明其具有免疫原性。结论 运用基因工程技术获得的表达产物为重组 Ｇ Ａ Ｄ６５ 抗原。这一抗原的获得，将有助于１ 型糖尿病的预测、预防、鉴别诊断以及免疫治疗等方面的研究。

Objective To getthe glutamic acid decarboxylase ( G A D65) antigen which can be used inexperimentalstudy and clinicalapplication . Methods c D N Afor G A D65 gene wasinsertedintothe expressionvector,and its expression in E.coli. D H5αwas induced . The molecular weight of expression product wasdetermined by S D S P A G Eand im munogenicity of expression product was ascertained by E L I S A. Results The sequence analysis confirmed that G A D65 gene had been correctly insertedinto p G E X 3 X. The molecularweightof the expression product was approximately 63 .23 K D, which was within the permitted error ( <8 % ) and similar to that of G A D65 antigen . Indirect E L I S Ashowed thatthe obvious color reaction appearedafterthe expression products were associated with the diabetic serum . It proved thatthe expression productshad expected im munogenicity . Conclusion The expression products obtained by the method of geneengineering are recombinant G A D65 antigen and may be used in experimentalstudy and clinicalapplication fordiabetes mellitus.