锌转运蛋白8在酿酒酵母中的重组表达及其抗原性分析

Expression of zinc transporter 8 in Saccharomyces cerevisiae and its antigenicity analysis

摘要:

锌转运蛋白8(zinc transporter 8,ZnT8)是Ⅰ型糖尿病的重要候选抗原，基于ZnT8所开发的自身抗体检测试剂盒可用于帮助诊断Ⅰ型糖尿病，相关产品已在欧美国家上市。目前，国内正在积极开发Ⅰ型糖尿病检测试剂盒，由于国内尚未建立活性ZnT8的重组生产体系，因此这一关键原料严重依赖进口。本研究主要利用酿酒酵母系统开展了ZnT8的重组表达研究。首先，设计了ZnT8的多种抗原形式，分别为C端单倍体蛋白(C)、C端二倍体(C-C)以及N端和C端串联体蛋白(N-C)，并使用酿酒酵母系统对上述蛋白进行了重组表达与纯化。随后，通过桥连法ELISA对纯化蛋白进行了抗原性测试，检测13位Ⅰ型糖尿病病人血清及16位健康志愿者血清后发现：C、N-C、C-C蛋白具有相似的检出率，分别为53.8%(7/13)、61.5%(8/13)及53.8%(7/13)；3组的特异性均为100%(16/16)；N-C蛋白相对其他2个蛋白而言，其在P3、P4、P8阳性样本上的检测值提高90%以上，表明具有更好的血清抗体识别能力。最后，选取N-C蛋白做进一步的血清样本测试，并将测试结果用ROC曲线进行敏感性及特异性表征，结果表明，与进口金标抗原相比，本方法敏感性无明显差异，特异性有待提高。综上可见，本研究基于酿酒酵母表达生产的ZnT8 N-C串联体蛋白有潜力作为Ⅰ型糖尿病体外诊断试剂开发中的国产替代原料。

关键词:自身免疫;ELISA;Ⅰ型糖尿病;ZnT8

Abstract:

Zinc transporter 8 (ZnT8) is an important candidate antigen for type Ⅰ diabetes. The autoantibody detection kit based on ZnT8 can be used to help diagnose type Ⅰ diabetes, and the related products have been launched in Europe and the United States. Since the recombinant production system of active ZnT8 has not been established in China, this key raw material is heavily dependent on imports. We used Saccharomyces cerevisiae to carry out the recombinant expression of ZnT8. First, multiple antigenic forms of ZnT8 were designed as C-terminal haploid (C), C-terminal diploid (C-C), and N-terminal and C-terminal concatemers (N-C). The proteins were expressed, purified and tested for antigenicity by bridging-type ELISA. The serum of 13 patients with type Ⅰ diabetes and the serum of 16 healthy volunteers were detected. C, N-C, and C-C proteins had similar detection rates, which were 53.8% (7/13), 61.5% (8/13) and 53.8% (7/13). The specificity of the three groups was 100% (16/16). The detection value on positive samples P3, P4, and P8 increased by more than 90%, indicating better serum antibody recognition ability. Finally, N-C protein was selected for further serum sample testing, and the test results were characterized by receiver operating characteristic (ROC) curve for sensitivity and specificity. Compared with imported gold standard antigen, the sensitivity was 76.9% (10/13) and the specificity was 87.5% (14/16). There was no significant difference in the sensitivity of the method, but the specificity needed to be improved. In conclusion, the ZnT8 N-terminal and C-terminal concatemer protein developed based on S. cerevisiae expression system is expected to be a key alternative raw material in the development of in vitro diagnostic reagents for type Ⅰ diabetes.

Key words:autoimmunity;ELISA;T1DM;ZnT8