title: Expression, localization, and clinical application of the RNA binding domain of U1-70kD in HEp-2 cells.
abstract: OBJECTIVE
  To develop an improved substrate for indirect immunofluorescent test (IIF) to detect anti-U1-70kD autoantibodies.
  
  
  METHODS
  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.
  
  
  RESULTS
  (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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Shun-le
authors.initials: SL
authors.lastname: Chen
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Shen
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xue
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Ye
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Chun-de
authors.initials: CD
authors.lastname: Bao
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Yue-ying
authors.initials: YY
authors.lastname: Gu
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Chong-zhao
authors.initials: CZ
authors.lastname: Yu
authors.affiliation: Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China lu_liangjing@medmail.com.cn.
authors.forename: Liang-jing
authors.initials: LJ
authors.lastname: Lu
mesh_terms: D000941:Antigens / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D045744:Cell Line, Tumor; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
chemical_list: D000941:Antigens; D001323:Autoantibodies; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; D049452:Green Fluorescent Proteins
keywords: HEp-2 cells; U1-70kD; antigenic localization; indirect immunofluoscence; transfection