OBJECTIVE: To prepare the specific antibodies against exon 2 and exon 3 of human tau protein. METHODS: Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA. RESULTS: Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 10(3). Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes. CONCLUSION: Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.
OBJECTIVE: To prepare the specific antibodies against exon 2 and exon 3 of human tau protein. METHODS: Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA. RESULTS: Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 10(3). Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes. CONCLUSION: Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.