IA-2基因的克隆及原核表达研究

目的 应用基因获取足量且具有免疫活性IA－2重组蛋白，为建立IA－2抗体检测方法奠定基础。方法 通过逆转录聚合酶链反应（RT－PCR）法从小鼠脑组织RNA中扩增编码IA－2胸内结构域的cDNA，构建表达型重组质粒，以DNA序列分析确证后，在大肠杆菌中表达，用亲和层析纯化融合目的蛋白，并以斑点印迹技术（Dot blot)鉴定其免疫原性。结果 所获特异CR产物正确重组至Pimpoint Xa-1表达载体中。经异丙基硫代半乳糖苷诱导的原核表达及亲和层析得到了纯度较高、相对分子质量约56000的融合目的蛋白，表达量约1mg蛋白／升菌液，且表达产物与谷氨酸脱羧酶抗体阳性的糖尿病患者血清及健康对照血清发生的显色反应差异有显著性（P＜0.05)。结论 原核表达的IA－2融合蛋白具有免疫学活性，可用用于糖尿病患者血清中相关抗体的检测。

Molecular cloning and prokaryotic expression of IA-2 gene

Objective To get sufficient IA 2 recombinant protein with immunoreactivity for the assay of IA 2 autoantibodies. Methods The gene encoding the intracellular domain of IA 2 was amplified from neonatal mouse brain tissue RNA by RT PCR, and was subcloned into Pinpoint Xa 1 T vector to construct recombinant expression plasmid. After sequence and open reading frame confirmed by DNA sequencing, the expression of target DNA in E. coli was induced. By using avidin resin, the biotinylated fusion protein (molecular weight about 56000) was affinity purified under non denaturing conditions. Thereafter, its immunogenicity was identified by dot blot using the sera of positive glutamic acid decarboxylase antibody from 10 diabetic patients as compared with the sera from healthy controls. Results The PCR products were correctly inserted into restriction sites of expression vector. The overall yield of purified recombinant protein was about 1 mg/L of bacteria culture. The reaction of IA 2 recombinant protein with GAD antibodies positive sera of diabetic patients was significantly different from those with sera of healthy controls. Conclusion The expressed IA 2 fusion protein shows expected immunoreactivity and will be used in further researches on type 1 diabetes.