Molecular cloning of cDNAs expressing SS-B/La protein.
Using serum from a patient with Sjögren's syndrome containing a high titer of anti-SS-B/La antibody, cDNA clones (a representative clone was called pA158) were isolated from a human fibroblast cDNA library in lambda gt11 expression vector. After subcloning of pA158 cDNA into an expression plasmid vector pEX-2, a large amount of the recombinant fusion protein with cro-beta-galactosidase (called pA158EX) was obtained in E. coli culture containing the recombinant pEX-2. Antibodies against pA158EX were purified from the patient serum by Sepharose 4B conjugated with the purified pA158EX protein. Immunofluorescent staining of HEp-2 cells with the anti-pA158EX antibodies showed a speckled nuclear staining. In immunoblot analysis, the anti-pA158EX antibodies reacted with 50 kDa protein that was compatible with SS-B/La protein. Immunoprecipitation of leukocyte lysate with the anti-pA158EX antibodies and the following RNA analysis showed that the antibody precipitated Y5 RNA. These findings indicate pA158 is a cDNA for SS-B/La protein. The purified fusion protein was used for enzyme-linked immunosorbent assay (ELISA). Optical density values of anti-SS-B positive sera were high, but those of anti-SS-B negative sera and healthy donor sera were low. In the Northern blot using human RNA and pA158 cDNA, a single band about 1.8 kb was recognized. A full-length cDNA was further obtained by screening of pcD library using pA158 cDNA as a probe.
Kohsaka
H
H
Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Japan.
Yamamoto
K
K
Fujii
H
H
Miyasaka
N
N
Miura
H
H
Tanaka
Y
Y
Nishioka
K
K
Miyamoto
T
T
D000906:Antibodies / Q000032:analysis / Q000276:immunology; D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear; D012859:Sjogren's Syndrome / Q000097:blood / Q000276:immunology; D000097585:SS-B Antigen
D000906:Antibodies; D001324:Autoantigens; D004247:DNA; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D000097585:SS-B Antigen