Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery
Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery
复制标题
DOI：
10.1073/pnas.0408782102
复制
发表时间：
2005-01-18
影响因子：
11.1
通讯作者：
Felgner, PL
中科院分区：
文献类型：
--
作者：
Davies, DH;Liang, XW;Felgner, PL
尽管许多人类病原体的基因组序列越来越多，但完整的proteornes的生产仍然处于瓶颈状态。为了满足这一需求，一个高通量PCR重组克隆和表达平台已经开发出来，允许数百个基因通过使用普通的实验室程序批量处理，而不需要机器人技术。该方法通过使用基因特异性引物对每个预测的ORF进行高通量扩增，然后在体内同源重组成T7表达载体。这些蛋白在大肠杆菌为基础的无细胞体外转录/翻译系统中表达，含有表达蛋白的粗反应不经纯化直接打印到硝化纤维微阵列上。蛋白质微阵列可用于确定接种疫苗或受感染的人和动物的完整抗原特异性体液免疫反应谱。通过克隆、表达和打印由185个单独的病毒蛋白组成的牛痘病毒蛋白质组，验证了该系统。该芯片用于测定牛痘病毒免疫的人、灵长类动物和小鼠血清中的Ab谱。人血清具有高滴度的抗e。大肠杆菌抗体需要阻断以揭示痘苗特异性反应。幼稚的人对13种抗原的亚群表现出反应性，这些抗原与痘苗免疫无关。幼稚的老鼠和灵长类动物缺乏这种背景反应。尽管牛痘免疫后抗原的一个共同子集是反应性的，但三个物种之间的特异性谱不同。这些结果验证了该平台是一种快速全面扫描接种疫苗或感染人类和动物体液免疫的方法。
Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteornes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.