Extracting And Expressing Amelogenin And It’s Functional Fragments LRAP And TRAP

Objective: Extracting full-length amelogenin and it’sfunctional fragments LRAP and TRAP to reconstruct and express in prokaryotes.Methods: This study was consisted of two parts:Experiment 1: Total RNA was extracted from the tooth germs of mandibular third molar which was in enamel developmental stages, the amelogenin c DNA was obtained from the total RNA through RT-PCR.The segments were inserted into p MD19-T vectorand the result plasmids were transformed into E.coli Top10.This positive clone was sequenced to identification. The identified homo amelogeninc DNA were served as a template to construct LRAP by overlap extension PCR and to amplify TRAP. Sequencingof LRAP and TRAP after cloningwere identified.Experiment 2: The homo amelogenin, LRAP and TRAP were inserted into prokaryotic expression vector p ET-28a(+).The recombinant plasmids were transformed into E.coli BL21 plys and induced by IPTG. The expressed products were purified and used SDS-PAGE to identify. Because LRAP and TRAP were unexpressed, LRAP and TRAPwere insertedinto prokaryotic expression vector p Cold-SUMO again. The recombinant plasmids were transformed into E.coli BL21(DE3) Chaprone. The expressed products were digested by r TEV. The recombinant protein was purified on Ni-NTA resin and used SDS-PAGE to identify.Results: The sequence of plasmids p MD19-T-Amshowed that nucleotide sequence of amelogenin which cloned were consistent with NCBI record. The sequence ofplasmids p MD19-T-LRAP and p MD19-T-TRAP demonstrated that nucleotide sequence of LRAP and TRAP were consistent with those references appeared.After induction, new protein bands of p ET-28a(+)-Amwere observed on SDS-PAGE gel near 22 k Da but LRAP and TRAP were not.Afterpurification, new protein bands of p Cold-SUMO-LRAPand p Cold-SUMO-TRAP appeared on SDS-PAGE gel near 5-6k Da.Conclusion: Homo amelogenin, LRAP and TRAP can be cloned from total RNA of the tooth germs extract from mandibular third molars which is in enamel developmental stages.Human genes of amelogenin, LRAP and TRAPare successfully expressed in prokaryotic.