[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].
OBJECTIVE
To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity.


METHODS
Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA.


RESULTS
The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA.


CONCLUSIONS
The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.
Department of Urology, General Hospital of PLA, Beijing, China. yxtwy@163.com
Xiaotao
X
Yin
Wei
W
Wang
Renli
R
Tian
Yuanji
Y
Xu
Jinqi
J
Yan
Wei
W
Zhang
Jiangping
J
Gao
Jiyun
J
Yu
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D011993:Recombinant Fusion Proteins; D000077022:Survivin