TIA重组cDNA表达克隆血清抗原基因的识别

Identification of serological antigens by recombinant cDNA expression cloning for TIA

目的:应用重组cDNA表达克隆血清学鉴定技术(SEREX)筛选与识别短暂性脑缺血发作(TIA)血清相关性抗原基因.方法:采集19例TIA患者血清,免疫筛选人主动脉内皮细胞cDNA表达文库;对阳性靶抗原行单克隆扩增,经单克隆内切除后转换载体pBluescript SK(+)提取质粒;对包含cDNA插入目的基因片段的质粒进行DNA序列检测,通过NCBI-BLAST同源性检索,鉴定具有免疫原性的独立的TIA重组cDNA表达克隆血清抗原基因.结果:(1)SEREX筛选出172个重组cDNA阳性单克隆;(2)单克隆内切除后获得167个pBluescript质粒,酶切鉴定质粒中均包含有目的基因插入片段;(3)生物信息学分析:识别11个独立的重组cDNA表达克隆抗原基因.(4)基因功能解析:发现matrix metalloproteinase 1(MMP1)、chromobox homolog 1(CBX1)和chromobox homolog 5(CBX5)与TIA和脑梗死(CI)的发生、发展关系密切.结论:应用SEREX技术成功筛选及鉴定出TIA相关靶抗原基因,发现MMP1、CBX1和CBX5可能与短暂性脑缺血发作(TIA)和脑梗死(CI)发生发展有关.

Objective： For early diagnosis of transient ischemic attack （TIA）, TIA-associated antigens were screened recombinant cDNA expression cloning （SEREX）. Methods： TIA patient serum antigens recognized by IgG were screened by SEREX using a human aortic endothelial cell cDNA library. Positive monoclonal phage cDNA clones were converted to pBluescript phagemids by single-clone excision. The pBluescript plasmids containing inserted cDNAs were obtained and the cDNA inserts were sequenced and analyzed for homology with known genes or proteins using NCBI-BLAST. Results： 172 positive monoclonal phage cDNA clones were obtained by SEREX. Sequences of 167 cDNA insertions were searched for homology with identified genes from the public sequence database, and 11 independentcDNA clones were identified. Gene function analysis demonstrated that matrix metalloproteinase 1 （ MMP1 ）, chromobox homolog 1 （ CBX1 ） and chromobox homolog 5 （ CBX5 ） were associated with TIA and CI. Conclusion： SEREX screening identified 11 TIA-associated antigens, among which MMP1, CBX1, and CBX5 could be closely related to the development of TIA and CI.