kinectin抗体免疫荧光检测方法的建立与应用

Establishment of Immunofluorescence assay for detecting kinectin autoantibody based on kinectin-transfected HepG2 cell line

目的 探讨kinectin蛋白在真核细胞的定位 ,并建立kinectin抗体免疫荧光检测方法(IFA)。方法 从人喉癌上皮细胞 (HepG2 )抽提RNA ,用逆转录聚合酶链反应扩增kinectin编码区序列 ,并连接到真核表达载体pEGFP C2中 ;将构建的 pEGFP kinectin载体导入HepG2细胞中 ,在荧光显微镜下观察绿色荧光蛋白 (GFP)的荧光及其细胞定位。然后 ,用此转染细胞制成的底片 ,建立检测kinectin抗体的IFA。同时用IFA对 5 1名正常人及 4 6例白塞病 (BD)患者血清进行初步检测。结果经EcoRI单酶切和测序证实 ,成功地构建了表达kinectin的真核表达载体 ;pEGFP kinectin载体转染的细胞经 3次克隆筛选后 ,10 0 %的细胞胞浆内发出特征性荧光。用kinectin抗体阳性血清染色 ,也得到类似特征的荧光。IFA检测显示 ,BD患者kinectin抗体的阳性率为 32 7% ( 15 /4 6 ) ,正常人均为阴性。结论 构建kinectin表达的载体可在HepG2细胞中进行表达 ,并成功地用 pEGFP kinectin转染的HepG2细胞建立了检测kinectin抗体的IFA。

Objective To establish a immunofluorescence assay for detecting kinectin autoantibody using kinectin transfected HepG2 cell line Methods RNA was purified from HepG2 line culture, kinectin sequence was amplified by RT PCR and cloned into the eukaryotic expression vector pEGFP C2, then transfected into Hep 2 cell The Hep 2 cell transfected with pEGFP kinectin was detected for green fluorescence at 488 nm of ultraviolet light A immunofluorescence assay (IFA) was set up using transfected cell as substrate for detecting the kinectin autoantibody in 46 behcet's disease and 51 normal controls Results A kinectin expressing eukaryotic vector, pEGFP kinectin, was successfully constructed and confirmed by DNA sequencing and single enzyme (EcoR I) digestion analysis After three times of subcloning and screening, 100% of the Hep 2 cell transfected with the pEGFP kinectin vector, excited at 488 nm of ultraviolet light gave a green fluorescence pattern characterized by a nucleus surrounding cytoplasmic staining A similar characteristics staining pattern was found in the transfected Hep 2 cell using Behcet' patient serum and can be diminished by recombinant kiinectin antigen Using the IFA, the positive rate of kinectin was 32 7% (15/46) in behcet's disease patients The positive results was not found in normal controls Conclusion The human kinectin gene was successfully cloned and expressed in Eukaryotic cell A IFA for detecting kinectin antibody was successfully established