用体外表达的血小板膜蛋白GPⅢa偶联Luminex微球检测HPA-1a抗体  

Application of recombinant GPⅢ a combined Luminex beads for the detection of HPA-la antibody

作　　者：陶苏丹[1] 刘瑛[1] 和艳敏[1] 应燕玲[1] 何吉[1] 朱发明[1] 

机构地区：[1]浙江省血液中心,卫生部血液安全研究重点实验室,浙江省血液安全研究重点实验室,杭州310006

出　　处：《中华医学遗传学杂志》2017年第1期40-44,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金面上项目（81371905、81570170）;浙江省医药卫生科学研究基金（WKJ-ZJ-1509、2014KYB066、2016RCB006、2011ZDA005）

摘　　要：目的拟利用体外克隆表达的GPⅢa蛋白作为HPA—1a抗原与LuminexxMAP微球相连，建立和评估用Luminex微球技术检测血清中抗HPA—1a的效果。方法扩增并克隆GPⅢa蛋白的编码基因ITGB3至真核表达载体，转染中国仓鼠卵巢癌细胞株。利用G418筛选稳定表达细胞株，体外获取并鉴定相应的特异性膜糖蛋白GPⅢa。将重组蛋白通过表面游离氨基和Luminex xMAP微球表面激活的羧基相偶联，再利用偶联后的微球与待测血清反应。利用PE-抗人IgG进行显色，在Luminex-100仪上读取数据并判断结果。结果ITGB3基因测序为HPA-1aa，将克隆后的GPⅢa重组蛋白偶联Luminex xMAP微球，Luminex微球法检测HPA-1a抗体的灵敏度为滴度1／32（抗体浓度3．125U／mL）。微球技术能特异性鉴别出待测样本的HPA—1a抗体，与MAIPA结果一致，而且与HLA抗体、ABO抗体以及其他HPA血小板系统抗体（HPA-3a、HPA_5b）均不发生交叉反应，说明建用立的Luminex微球技术来检测血小板抗体是可行的。结论成功获取了重组表达的血小板膜蛋白GPⅢ3，并初步建立了Luminex微球检测HPA-1a抗体方法。Objective To generate recombinant GPⅢ a as an alternative source for HPA-la antigen and combine it with Luminex xMAP beads for the detection of HPA-la-specific alloantibody. Methods The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer. Results DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GP Ⅲ a was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 （3. 125 U/mL）. The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens （MAIPA） technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies （HPA-3a and HPA-Sb）. The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible. Conclusion Recombinant GPⅢ a was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

关 键 词：Luminex微球技术 GPⅢa体外重组表达 抗HPA—1a 

分 类 号：R457.1[医药卫生—治疗学]