title: [Prokaryotic expression of human GITRaa27-165 and preparation of its polyclonal antibody].
abstract: OBJECTIVE
  To express and purify the extracellular region of human glucocorticoid-induced tumor necrosis factor (hGITR(aa27-165)), and to prepare and identify the polyclonal antibody (pAb) against this fusion protein.
  
  
  METHODS
  The 429 bp DNA sequence of hGITR(aa27-165) was obtained from pGEM T-hGITR by PCR and then it was inserted into pQE30 plasmid to construct the prokaryotic expression plasmid pQE30-hGITR(aa27-165). pQE30-hGITR(aa27-165) was transformed into E.coli. The target fusion protein was expressed with the induction of Isopropyl beta-D-1-thiogalacto- pyranoside (IPTG) and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hGITR(aa27-165) was obtained from the rabbits immunized with hGITR(aa27-165). The titer of pAb was detected by ELISA and the specificity of pAb was identified by Western blot.
  
  
  RESULTS
  The prokaryotic expression plasmid pQE30-GITR(aa27-165) was constructed successfully. The culture condition in which the target fusion protein was highly expressed was found out: the optimal concentration of IPTG was 0.5 mmol/L, the culture temperature was 30 degree centigrade and the culture time was 6 h. The GITR(aa27-165) fusion protein was effectively expressed in E.coli as inclusion body. Soluble protein was obtained through denaturation and refolding procedure. The fusion protein was purified by Profinity IMAC Ni-Charged Resin affinity column with above 90% purity. The anti-GITR(aa27-165) pAb was prepared by immunizing the rabbits with the purified target fusion protein. ELISA showed the titer of pAb was 1:1.6 x 10(5). Western blot analysis confirmed anti-GITR(aa27-165) pAb was of good specificity.
  
  
  CONCLUSIONS
  The fusion protein hGITR(aa27-165) with high purity has been obtained and the anti-hGITR(aa27-165) pAb with high titer and good specificity has been prepared. Our study may be conductive to further research into the molecular mechanism of action between human GITR and GITRL.
authors.affiliation: Department of Immunology and Laboratory Immunology, School of Medical Technology, Zhenjiang 212013, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Tong
authors.forename: Sheng-jun
authors.initials: SJ
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Chao-ming
authors.initials: CM
authors.lastname: Mao
authors.forename: Yun-liang
authors.initials: YL
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.forename: Zheng-jun
authors.initials: ZJ
authors.lastname: Hu
authors.forename: Di-yuan
authors.initials: DY
authors.lastname: Zhi
authors.forename: Hua-Xi
authors.initials: HX
authors.lastname: Xu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D053450:Glucocorticoid-Induced TNFR-Related Protein; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000906:Antibodies; D053450:Glucocorticoid-Induced TNFR-Related Protein; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C491405:TNFRSF18 protein, human