title: [Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis].
abstract: OBJECTIVE
  To clone the peptidylarginine deiminase(PAD) gene of Porphyromonas gingivalis(P.gingivalis) and to be expressed in E. coli under the best conditions.
  
  
  METHODS
  With P.gingivalis strains ATCC33277 genomic DNA as a template, PCR was applied to obtain gene PAD which was then inserted into linear cloning vector PMD-18-T to construct clone recon. Recombinant PMD18-T-PAD was cloned and analyzed with PCR and restriction endonuclease,and PET-28a expression vector was digested by Xhol and Ncol,their products were linked to construct expression plasmid PET-28a-PAD under certain connection system. The recombinant expression plasmid PET-28a-PAD which had been confirmed correctly was transformed to E. coli competent cells BL21 and induce the expression of PAD with IPTG of different density and time. With His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot.
  
  
  RESULTS
  DNA sequencing showed that the fragment was same as the sequence published in NCBI. Under the condition of 37 degrees centigrade, 0.5mmol/L IPTG, 250r/min shaking for 6 hours, the PAD could be highly expressed.
  
  
  CONCLUSIONS
  The PAD is successfully cloned and expressed in E. coli which can be further uesd to study the immunity of PAD and the homologues antibody preparation.
authors: Research Center of Stomatology, Xi'an Jiaotong University. Xi'an, Shanxi Province, China. drliang@mail.xjtu.edu.cn
authors: Ang
authors: A
authors: Li
authors: Chun-hui
authors: CH
authors: Zhu
authors: Jian-feng
authors: JF
authors: Shi
authors: Hong
authors: H
authors: Wei
authors: Jin
authors: J
authors: Liu
authors: Jian-zhong
authors: JZ
authors: Gou
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli; D005822:Genetic Vectors; D006867:Hydrolases; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis; D000076342:Protein-Arginine Deiminases
chemical_list: D006867:Hydrolases; D000076342:Protein-Arginine Deiminases