ABSTRACT
Objective To detect the possible epitopes of 52 000 dalton Ro/SSA autoantigen and selectively clone the antigen fragment containing the leucine zipper motif. Methods The structure of 52 000 dalton Ro/SSA autoantigen was analyzed by protein structure analysis system. The cDNA encoding the antigen fragments from amino acid (aa) 119 to 264 was amplified from cDNA first strands of human heart by polymerase chain reaction (PCR). The PCR product was inserted into the vector pMTY4, and transfected E. coli pop 2136 for fusion protein production. Results Computer analysis showed that the 52 000 dalton Ro/SSA antigen had alpha helixes in its structure, and had good antigenicity, moderate flexibility and poor surface probability. The PCR product was 440bp in length. The recombinant fusion protein was 28 000 dalton. DNA sequencing proved the accuracy of the research. Conclusion The 52 000 dalton Ro/SSA autoantigen has good antigenicity. The cloning of the antigen fragment will help to elucidate the possible role of leucine zipper motif in forming the epitope of the antigen.
ABSTRACT
Objective To investigate the role of Ro52 antigen fragment from amino acid (AA) 119 to 264 that contains leucine zipper motif in forming the epitopes of Ro52 autoantigen. Methods cDNA encoding Ro52 antigen fragment from aa 119 to 264 was amplified by PCR from human heart cDNA first strand. The obtained cDNA fragment was inserted into vector pMTY4 and transformed into E. coli pop2136 for fusion protein expression. The recombinant fusion protein was then purified, and the antigenicity was determined by immunoblotting. Results A 28 000 Dalton fusion protein was obtained. It was highly expressed in host E. coli pop2136. 61.90% of the anti-full-length Ro52 positive sera reacted with the fusion protein in immunoblotting. Conclusion The results suggest that the antigen fragment from the 119 AA to 264 AA that contains the leucine zipper motif represents an important epitope region in Ro52 autoantigen.