多聚抗原肽微阵列分析平台的建立与初步应用

基于多肽设计合成技术建立了一个快速、高通量、自动化的多聚抗原肽微阵列分析平台.选取人巨细胞病毒(HCMV)的包膜糖蛋白B和被膜碱性磷酸蛋白PP150,幽门螺杆菌(Helicobacterpylori,Hp)尿素酶(Ure)β亚基为靶蛋白,分析筛选出优势线性表位序列,Fmoc法固相合成上述线形表位的多聚抗原肽(MAPs),高效液相色谱仪(HPLC)纯化后,用机器人点样仪按一定的矩阵排列形式点印至硝酸纤维素膜上,2%的小牛血清封闭,塑料壳体封装制备成MAPs微阵列成品.随机抽样经质控血清鉴定后用于随机人群血清试验并与ELISA检测结果进行比较.筛选、合成并鉴定出4条MAPs.用该MAPs微阵列检测的Hp和HCMV阳性及阴性质控血清结果均与质控血清情况相符,120份随机血清检测结果与用重组抗原和病原微生物裂解物抗原包被的ELISA法检测结果相比具有较好的一致性,Ure-1、Ure-2和PP150三种MAPs的灵敏度和特异性均大于90%.MAPs微阵列片间质控试验结果变异系数小于7%,示重复性良好.MAPs微阵列是一种快速、高通量、自动化的分析平台,该平台在预防性疫苗的开发和蛋白质组学的研究中具有较大的前景.

The Development and Preliminary Application of Multiple Antigenic Peptide Microarray Platform

A fast, high-throughput, automated multiple antigenic peptides microarray platform was established based on multiple peptide design and synthesis technology. Human cytomegalovirus(HCMV) glycoprotein B, phosphoprotein pp150 and Helicobacter pylori (Hp) urease (Ure) beta subunit were chosen as target protein. The advantageous linear epitope of these proteins was analyzed and screened. The multiple antigenic peptides(MAPs) containing the selected sequence were synthesized by Fmoc solid phase method,purified through high performance liquid chromatography(HPLC) and printed on nitrocellulose membrane in microarrays by computer-controlled high-speed robotics. The nitrocellulose membrane was blocked with 2% bovine serum albumin solution. The MAPs microarray was finished by assembling the membrane with plastic outer shell. Some microarrays were selected at random for quality control identified by control sera and compared with ELISA method. 4 MAPs were screened out, synthesized and identified. The result of positive and negative sera of Hp and HCMV detected by MAPs microarray were consistent with the control sera. In the clinical trial of 120 random sera, the microarray performed almost equally with ELISA method using recombinant antigen and microbial lysate antigen. The sensitivity and specificity of Ure-1, Ure-2 and PP150 MAPs were higher than 90%. The CV were lower than 7% among microarrays showing a good repeatability. It can be concluded that a MAPs microarray analytical platform which has a vast application prospect in assistant design of peptide vaccine was primarily established.