title: [Cloning and expression of the prokaryotic expression vectors of phytoplasma immunodominant membrane protein A and preparation of its antiserum].
abstract: OBJECTIVE
  To construct the prokaryotic expression vector of phytoplasma immunodominant membrane protein A (IdpA) in prokaryotic cell, express and purify the IdpA and prepare its antiserum.
  
  
  METHODS
  With the recombinant plasmid pMD18-T-IdpA as templates, IdpA gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a(+) by endonuclease reaction and T4 DNA ligase reaction. Then the recombinant plasmid pET-28a(+)-IdpA was transformed into E.coli BL21 (DE3). After confirmed by PCR and double enzyme digestion, the recombinant protein IdpA was expressed under IPTG induction and purified. The purified product was used to immunize BALB/c mice to prepare its antiserum. IdpA-specific mouse antiserum was identified by ELISA and Westerrn blotting.
  
  
  RESULTS
  The prokaryotic vectors of pET-28a(+)-IdpA were constructed successfully and the recombinant protein IdpA was induced to express stably in the E.coli BL21. The purity of IdpA was up to over 90%. In the BALB/c mice immunized by the purified IdpA, the titre of IdpA-specific antiserum was as high as 1:320 000.
  
  
  CONCLUSIONS
  The recombinant protein IdpA was expressed successfully in E.coli and the IdpA-specific antiserum was prepared.
authors.affiliation: College of Life Science, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Liang
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhang
authors.forename: Haiquan
authors.initials: H
authors.lastname: Zhao
authors.forename: Zhongjian
authors.initials: Z
authors.lastname: Liu
authors.forename: Huanliang
authors.initials: H
authors.lastname: Luo
authors.forename: Yanxing
authors.initials: Y
authors.lastname: Lin
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D010957:Plasmids
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007106:Immune Sera; C435308:immunodominant membrane protein A, phytoplasma