CAJ | 학술논문

目的　应用基因工程技术获取足量且具有免疫活性的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胞内结构域的cDNA，构建表达型重组质粒，经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PCR产物正确重组至Pinpoint 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56　　000的融合目的蛋白，表达量约1 mg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，可用于糖尿病患者血清中相关抗体的检测。
목적　응용기인공정기술획취족량차구유면역활성적IA-2중조단백，위건립IA-2항체검측방법전정기출。방법　통과역전록취합매련반응(RT-PCR)법종소서뇌조직RNA중확증편마IA-2포내결구역적cDNA，구건표체형중조질립，경DNA서렬분석학증후，재대장간균중표체，용친화층석순화융합목적단백，병이반점인적기술(Dot blot)감정기면역원성。결과　소획특이PCR산물정학중조지Pinpoint Xa-1표체재체중。경이병기류대반유당감유도적원핵표체급친화층석득도료순도교고、상대분자질량약56　　000적융합목적단백，표체량약1 mg단백/승균액，차표체산물여곡　안산탈최매항체양성적당뇨병환자혈청급건강대조혈　청발생적현색반응차이유현저성(P＜0.05)。결론　원핵표체적IA-2융합단백구유면역학활성，가용우당뇨병환자혈청중상관항체적검측。
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 56　　000) 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.