Expression, localization, and clinical application of the RNA binding domain of U1-70kD in HEp-2 cells
| Autor: | Yang, Song, Shun-le, Chen, Nan, Shen, Feng, Xue, Ping, Ye, Chun-de, Bao, Yue-ying, Gu, Chong-zhao, Yu, Liang-jing, Lu |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Recombination
Genetic Recombinant Fusion Proteins Genetic Vectors Green Fluorescent Proteins Immunoblotting Transfection Polymerase Chain Reaction Protein Structure Tertiary Ribonucleoprotein U1 Small Nuclear Molecular Weight Cell Line Tumor Humans Antigens Fluorescent Antibody Technique Indirect Autoantibodies |
| Zdroj: | Annals of clinical and laboratory science. 45(2) |
| ISSN: | 1550-8080 |
| Popis: | To develop an improved substrate for indirect immunofluorescent test (IIF) to detect anti-U1-70kD autoantibodies.The RNA binding domain of U1-70kD (U1BD) complementary DNA was obtained from human larynx carcinoma cell line HEp-2 by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the mammalian expression vector pEGFP-C1. The recombinant plasmid pEGFP-U1BD was transfected into HEp-2 cells. Immunoblotting (IBT), confocal fluorescence microscopy, and IIF were used to confirm the expression, localization, and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected HEp-2 cells, which were then analyzed by IIF using human reference sera and compared with untransfected HEp-2 cells simultaneously.(1) The HEp-U1BD cells thus obtained retained their ability to express U1BD-GFP, which showed the antigenicity of U1BD with a characteristic phenotype in IIF. (2) Fifteen IBT-positive anti-U1-70kD sera presented with characteristic cytoplasmic staining on HEp-U1BD by IIF, but five sera without the 70kD reactive band in IBT were not found in the presence of HEp-U1BD pattern. Ten sera of healthy donors couldn't react with HEp-2 and HEp-U1BD at 1:80 attenuant degrees. (3) No differences in expression, localization, or morphology were observed when HEp-U1BD or HEp-2 interacted with the reference sera that could react with Ro/SSA, La/SSB, centromere, histone, and Scl-70 antigens in routine IIF test.HEp-U1BD cells kept the immunofluorescent properties of HEp-2 cells in an immunofluorescence anti-nuclear antibody (IFANA) test and could be potentially used as a substrate for routine IFANA detection. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|