To clone and express EC1 2 and EC3 4 epitopes of PVA (pemphigus vulgaris antigen, desmoglein 3) in order to diagnose pemphigus and study the relationship between epitopes of PVA and anti PVA antibody Methods RN...To clone and express EC1 2 and EC3 4 epitopes of PVA (pemphigus vulgaris antigen, desmoglein 3) in order to diagnose pemphigus and study the relationship between epitopes of PVA and anti PVA antibody Methods RNA was extracted from keratinocytes and the cDNA of epitopes EC1 2 and EC3 4 was synthesized by reverse transcription Amplified genes of EC1 2 and EC3 4 were inserted into the expression plasmid, PGEX 4T 1, and transformed into E coli BL21 by electric transduction Recombinant fusion proteins of EC1 2 and EC3 4 epitopes were expressed by IPTG induction These proteins were separated on SDS PAGE gels and electroblotted to nitrocellulose to detect the anti PVA antibody Results The sequences of cloned EC1 2 and EC3 4 genes were identical to the sequence registered in PC/GENE Expressed recombinant proteins reacted only to sera from patients with pemphigus vulgaris, not to sera from patients with bullous pemphigoid, systemic lupus erythematosus or normal persons Conclusions These recombinant proteins are very specific in antigenicity This may provide a new method for the diagnosis of pemphigus vulgaris (PV) or the differential diagnosis of other bullous cutaneous diseases via patient sera It is also helpful in understanding the relationship between adhesion molecules and the pathogenic mechanism of pemphigus vulgaris展开更多
基金This project was supported by the grants fam the Healh Administration of Shanghai(Na 98BR037) the National Natural Science Faundatian of China(Na 39870665).
文摘To clone and express EC1 2 and EC3 4 epitopes of PVA (pemphigus vulgaris antigen, desmoglein 3) in order to diagnose pemphigus and study the relationship between epitopes of PVA and anti PVA antibody Methods RNA was extracted from keratinocytes and the cDNA of epitopes EC1 2 and EC3 4 was synthesized by reverse transcription Amplified genes of EC1 2 and EC3 4 were inserted into the expression plasmid, PGEX 4T 1, and transformed into E coli BL21 by electric transduction Recombinant fusion proteins of EC1 2 and EC3 4 epitopes were expressed by IPTG induction These proteins were separated on SDS PAGE gels and electroblotted to nitrocellulose to detect the anti PVA antibody Results The sequences of cloned EC1 2 and EC3 4 genes were identical to the sequence registered in PC/GENE Expressed recombinant proteins reacted only to sera from patients with pemphigus vulgaris, not to sera from patients with bullous pemphigoid, systemic lupus erythematosus or normal persons Conclusions These recombinant proteins are very specific in antigenicity This may provide a new method for the diagnosis of pemphigus vulgaris (PV) or the differential diagnosis of other bullous cutaneous diseases via patient sera It is also helpful in understanding the relationship between adhesion molecules and the pathogenic mechanism of pemphigus vulgaris
