Yin Xiaotao, Wang Wei, Tian Renli, Xu Yuanji, Yan Jinqi, Zhang Wei, Gao Jiangping, Yu Jiyun
Department of Urology, General Hospital of PLA, Beijing, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Aug;29(8):877-81.
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.
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.
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.
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.
构建原核表达质粒pET28a-survivin，优化其在大肠杆菌中的重组蛋白表达条件，纯化survivin重组蛋白并鉴定其抗原性。
通过PCR扩增survivin cDNA片段，克隆至原核表达载体pET28a(+)中构建重组表达载体pET28a-survivin。将表达载体转化至BL21(DE3)中，用IPTG诱导融合蛋白survivin/His表达。通过镍亲和层析纯化融合蛋白。采用Western印迹法和ELISA鉴定纯化的survivin蛋白的抗原性。
经BamHI和HindIII酶切鉴定，重组表达载体构建成功。IPTG诱导获得了相对分子质量约为24 000的融合蛋白。SDS-PAGE分析显示纯化蛋白纯度达90%。Western印迹法和ELISA验证了survivin蛋白的抗原性。
成功构建了原核表达质粒pET28a-survivin，survivin蛋白在大肠杆菌中得到表达和纯化，纯化的survivin蛋白具有良好的抗原性。
