β_2糖蛋白I基因转染HEp-2细胞株中融合蛋白的表达及临床应用

Expression of fusion proteins in β_ 2GP I gene-transfected HEp-2 cells and its clinical application

目的 观察 β2 糖蛋白I( β2 GPI)基因转染的HEp 2细胞 (HEp β2 GPI)中 β2 GPI融合蛋白的过度表达。探讨以HEp β2 GPI为底物间接免疫荧光 (IIF)法检测抗β2 GPI抗体的可行性。方法 应用逆转录聚合酶链反应 (RT PCR)扩增人源 β2 GPIcDNA ,克隆入pEGFP C1 ,并转染HEp 2细胞。通过RT PCR、共焦荧光显微镜、免疫印迹法 (IBT)及IIF鉴定β2 GPI 绿色荧光蛋白 (GFP)融合蛋白的表达和抗原性。以转染细胞为底物IIF检测 1 9份疑诊继发性抗磷脂综合征 (APS)病人、1份原发性APS病人及 1 0份正常人血清的IgG β2 GPI,与同时进行的ELISA法检测IgG ACL、IgG β2 GPI的结果作比较。结果 ( 1 )获得的HEp β2 GPI细胞传十几代后仍具有较强的 β2 GPI GFP表达 ,融合蛋白保持 β2 GPI的抗原性 ,其IIF表型具有特征性。 ( 2 )IIF法测血清IgG β2 GPI的结果显示 :7份病人血清出现特征性免疫荧光表型 ,而正常人血清均无荧光染色。 3种方法的比较性研究显示IIF法测IgG β2 GPI与ELISA法测IgG β2 GPI的一致性程度最佳 (Kappa值 0 .886 )。 ( 3)HEp β2 GPI保持了原型HEp 2细胞检测抗核抗体 (ANA)的荧光特性 ,且检出 2例原型HEp 2细胞IIFANA是阴性的血清。结论 HEp β2 GPI转染细胞株可用于IIF检测抗 β2 GPI,而且仍可作?

Objective To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to β 2 glycoprotein. Methods Full length β 2GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT PCR and cloned into the mammalian expression vector pEGFP C1. The recombinant plasmid pEGFP β 2GP was transfected into HEp 2 cells. RT PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP). Serum specimens from 19 patients suspected as with secondary antiphopholopid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF IgG β 2GP1, ELISA IgG ACL, and ELISA IgG β 2GP I simultasneously. Results (1) The HEp β 2GP I cells thus obtained retained their ability of expression of β 2GP I GFP for more than ten generations. This β 2GP I GFP showed the antigenicity of β 2GP I with a characteristic feature. (2) Seven of the 20 serum specimens from APS patients showed characteristc immunofluorescent pattern. No serum specimen from normal persons showed immunofluorescent staining. The comparison of results of the three methods showed that the concordance between IIF IgG β 2GP I and ELISA IgG β 2GP I was the most perfect (Kappa= 0.886). (3) HEp β 2GP I retained the immunofluorescent property of HEp 2 cell. Conclusion As a new kind of substrate of IIF, β 2GP I transfectant can be used to detect anti β 2GP I antibodies. Transfeted HEp 2 cells keep the immunofluorescent property of HEp 2 cells in IFANA test and can be used as substrate for routine IFANA detection.