title: Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells.
abstract: OBJECTIVE
  To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti-Sm antibody.
  
  
  METHODS
  Full-length Smith protein D1(Sm-D1) 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-Sm-D1 was transfected into HEp-2 cells. The expression, localization and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected cells were confirmed by means of immunoblotting (IBT), confocal fluorescence microscopy and IIF analysis. Transfected HEp-2 cells were analyzed with reference serum and compared with untransfected HEp-2 cells by IIF.
  
  
  RESULTS
  Stable expression of the Sm-D1-GFP was maintained for more than ten generations. This Sm-D1-GFP showed the antigenicity of Sm-D1 with a characteristic phenotype in IIF.Six of 12 serum specimens from systemic lupus erythematosus contained both 29/28 and 13.5 kDa proteins and showed characteristic immunofluorescent patterns. The same phenomenon appeared in 3/6 serum samples which contained 29/28 kDa proteins only. Sera from 10 healthy donors did not react with HEp-Sm-D1 or HEp-2 at 1:80 attenuant degrees. No alteration in expression, localization and morphology was observed when HEp-Sm-D1 or HEp-2 interacted with the reference sera which could react with Ro/SSA, La/SSB, β2GP1, centromere, histone, and Scl-70 antibodies in routine IIF tests.
  
  
  CONCLUSIONS
  As a new kind of substrate of IIF, HEp-Sm-D1 can be used to detect anti-Sm antibodies. Transfected HEp-2 cells keep the immunofluorescent property of HEp-2 cells in immunofluorescence anti-nuclear antibody (IFANA) test and could potentially be used as substrate for routine IFANA detection.
authors: Department of Rheumatology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors: Su-li
authors: SL
authors: Wang
authors: Fang-fang
authors: FF
authors: Wang
authors: Shun-le
authors: SL
authors: Chen
authors: Nan
authors: N
authors: Shen
authors: Feng
authors: F
authors: Xue
authors: Ping
authors: P
authors: Ye
authors: Chun-de
authors: CD
authors: Bao
authors: Yue-ying
authors: YY
authors: Gu
authors: Chong-zhao
authors: CZ
authors: Yu
authors: Alisa
authors: A
authors: Wilson
authors: Daniel J
authors: DJ
authors: Wallace
authors: Michael H
authors: MH
authors: Weisman
authors: Liang-jing
authors: LJ
authors: Lu
mesh_terms: D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000097:blood; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D000939:Epitopes; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000378:metabolism*; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D055517:snRNP Core Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D000939:Epitopes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D055517:snRNP Core Proteins; D049452:Green Fluorescent Proteins
keywords: HEp‐2 cells; Sm‐D1; antigenic localization; indirect immunofluorescent technique; transfection