DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.48120#0020
2 Molecular and Cell Biology of Autoantibodies and Autoimmunity
Cloning of a cDNA-Fragment Coding for an Epitope Recognized by
Anti-PM/Scl-Autoantibodies
M. Bliithner1, E. Genth2, EA. Bautz'
1 Institute of Molecular Genetics, University of Heidelberg, Im Neuenheimer Feld 230,
D-6900 Heidelberg, FRG
2Rheumaklinik und Rheumaforschungsinstitut, D-5100 Aachen, FRG
Sera from patients suffering from Polymyositis-/Scleroderma-Overlap Syn-
drome (PM/Scl) recognize two major nucleolar proteins of 95 and 75 kd in
Western blots.
Human HeLa cDNA libraries constructed in Agtll were screened using affinity
purified anti 95 kd antibodies. Initial screening of 2.5 x 105 recombinants yielded
four putative cDNA clones coding for a 20 kd fragment of the 95 kd antigen,
which on further analysis were shown to be identical.
Antibodies eluted from the fusionprotein recognized the 95 kd antigen in Western
blot analysis and gave nucleolar staining in immunofluorescence.
EcoRI digestion of the recombinant DNA yielded a 551 bp fragment, which
was subcloned into Bluescript vector. Subsequent doublestrand sequencing ac-
cording to the dideoxy chain termination method showed one single PvuII restric-
tion site at the position 240. To verify this sequence, the EcoRI/PvuII-fragments
were subcloned into M Bmp 18 and M13mpl9 and sequenced independently from
both directions. A single open reading frame of the 551 bp fragment could be
verified by subcloning this fragment into expression plasmids pUR 290, pUR 291
and pUR 292 in both orientations. The existence of the antigenic sites of the
respective fusionproteins was probed by Western blotting with the PM/Scl-serum
used originally for the screening procedure. Only the fusionprotein expressed in
the correct orientation and reading frame gave a positive immunoreaction in
Western blot analysis. All eight PM/Scl-sera tested, including a PM/Scl-reference
serum reacted with this fusionprotein, suggesting, that we have cloned a major
epitope of the 95 kd PM/Scl-autoantigen.
Cloning of a cDNA-Fragment Coding for an Epitope Recognized by
Anti-PM/Scl-Autoantibodies
M. Bliithner1, E. Genth2, EA. Bautz'
1 Institute of Molecular Genetics, University of Heidelberg, Im Neuenheimer Feld 230,
D-6900 Heidelberg, FRG
2Rheumaklinik und Rheumaforschungsinstitut, D-5100 Aachen, FRG
Sera from patients suffering from Polymyositis-/Scleroderma-Overlap Syn-
drome (PM/Scl) recognize two major nucleolar proteins of 95 and 75 kd in
Western blots.
Human HeLa cDNA libraries constructed in Agtll were screened using affinity
purified anti 95 kd antibodies. Initial screening of 2.5 x 105 recombinants yielded
four putative cDNA clones coding for a 20 kd fragment of the 95 kd antigen,
which on further analysis were shown to be identical.
Antibodies eluted from the fusionprotein recognized the 95 kd antigen in Western
blot analysis and gave nucleolar staining in immunofluorescence.
EcoRI digestion of the recombinant DNA yielded a 551 bp fragment, which
was subcloned into Bluescript vector. Subsequent doublestrand sequencing ac-
cording to the dideoxy chain termination method showed one single PvuII restric-
tion site at the position 240. To verify this sequence, the EcoRI/PvuII-fragments
were subcloned into M Bmp 18 and M13mpl9 and sequenced independently from
both directions. A single open reading frame of the 551 bp fragment could be
verified by subcloning this fragment into expression plasmids pUR 290, pUR 291
and pUR 292 in both orientations. The existence of the antigenic sites of the
respective fusionproteins was probed by Western blotting with the PM/Scl-serum
used originally for the screening procedure. Only the fusionprotein expressed in
the correct orientation and reading frame gave a positive immunoreaction in
Western blot analysis. All eight PM/Scl-sera tested, including a PM/Scl-reference
serum reacted with this fusionprotein, suggesting, that we have cloned a major
epitope of the 95 kd PM/Scl-autoantigen.