HPA系统多态性位点附近的血小板膜糖蛋白基因新突变点的功能研究

Mutations in platelet membrane glycoprotein genes could lead to function alternation of membrane glycoprotein and associate with human platelet antigen systems (HPAs). This project will research for the relationship of mutations in platelet membrane glycoprotein genes and their function, which was based on our previously studies and the samples of the individuals with new mutations of platelet membrane glycoprotein genes which were found in our laboratory. Construction of recombinant expression vectors of GP1BA 517-525delAAC,ITGA2B 2722C>T,ITGA2 2474T>G, ITGB3(1333G>A、1476G>A、1813G>A) from mutation samples will perform, and the recombinant plasmids will transfect into the eukaryote cells and perform transient and stable expression analysis in vitro. The recombinant proteins will purify and analyze for its function. More techniques will be used in this research, including recombination and expression in vitro, immunofluorescence assay, activities determination of platelet glycoprotein and HPA antibody screening, which will clarity the effects of mutations in platelet glycoprotein genes on function of glycoprotein biosynthesis, activity and antigen expression. The recombinant protein expression system for the human platelet antigen will be established. It will explore the relationship of mutations in genes of platelet glycoprotein and human platelet antigen systems. The research will clarity the effect of the mutations on the epitope expression of nearly HPAs polymorphism in conformational space and the accuracy of HPA antibodies screening. In totally, it will clarify the function of mutations of the platelet glycoprotein genes which located nearly to human platelet antigen polymorphism sites in the project and this study will help to improve the blood safety.

血小板膜糖蛋白基因突变导致膜蛋白功能改变，与血小板同种抗原密切相关。本项目拟在课题组前期研究基础上，利用申请者实验室发现的血小板膜糖蛋白基因变异样本，开展具有自主知识产权的基因突变与功能研究。通过基因重组技术构建GP1BA 517-525delAAC,ITGA2B 2722C>T,ITGA2 2474T>G,ITGB3(1333G>A、1476G>A、1813G>A)突变的表达质粒进行体外表达研究，对重组蛋白进行纯化和功能分析，综合利用重组表达、免疫荧光、蛋白活性测定和抗体筛选等技术，阐明突变基因对相关糖蛋白合成、活性、抗原表达等功能的影响。拟构建HPA表达体系获取相应的特异蛋白，阐明突变点基因编码抗原与HPA系统的相关性，确定其在空间构象中对邻近的HPA系统表位表达的影响以及对抗体检测准确性的影响情况。项目将阐明HPA多态性位点附近的血小板糖蛋白基因突变点的功能作用，有利于提升血液安全。

人血小板同种抗原系统（HPA）不相合可引起血小板输注无效、新生儿血小板减少性紫癜等疾病。HPA系统的差异取决于血小板膜糖蛋白基因序列的多态性， 已发现HPA多态性位点附近存在其他的变异，但这些变异对HPA的影响尚不清楚。本项目通过基因重组技术构建了GP1BA 517-525delAAC,ITGA2B 2722C>T,ITGA2 2474T>G,ITGB3(1333G>A、1476G>A、1813G>A)突变表达质粒、稳定表达体系。以对应的野生型糖蛋白表达量作为100%，ITGA2B c.2722C>T、GP1BA c.517-525delAAC、ITGA2 c.2474T>G、ITGB3 c.1333G>A、ITGB3 c.1476G>A、ITGB3 c.1813G>A表达量分别为67%、63%、76%、104%、37%和89%。成功利用重组蛋白构建液相芯片技术检测HPA-1a抗体，检测4例阳性对照、8例阴性对照结果符合预期，未发现仅与突变蛋白结合的HPA抗体。选择GPIIb/IIIa抗体（P2）, GPIa/IIa抗体(Gi9)、GPIb/IX(AK2)、CD109 (W7/C5)抗体分别偶联微球，建立了全部HPA系统抗体的Luminex检测方法，标准品、ISBT第18、19届国际血小板协作组样品，检测结果全部符合预期。发现ITGB3 c.1476G>A终止突变可导致ITGB3基因的转录水平降低，影响GPⅢa蛋白合成及CD61抗原的表达水平。系统建立了HPA 1-28系统的基因多重扩增和测序方法，获取了汉族人群HPA 1-28系统遗传数据，并分析了错配概率和建库标本估算值，并为临床血小板输注无效提供了配合性解决方法。项目系统明确了实验室发现的临近HPA多态性位点的糖蛋白基因突变点的功能情况，这将有助于提升对HPA抗原系统的认知；建立的HPA抗体检测方法，已应用于血小板输注无效、新生儿血小板减少性紫癜等的实验室诊断，可提升血液安全。