Aim: 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.
Conclusion: 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.