[Prokaryotic expression of human GITRaa27-165 and preparation of its polyclonal antibody].
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.
Tong
Jia
J
Department of Immunology and Laboratory Immunology, School of Medical Technology, Zhenjiang 212013, China.
Wang
Sheng-jun
SJ
Chen
Jun
J
Mao
Chao-ming
CM
Yang
Yun-liang
YL
Yang
Min
M
Hu
Zheng-jun
ZJ
Zhi
Di-yuan
DY
Xu
Hua-Xi
HX
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*
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