Cloning of survivin-an inhibitor of apoptosis proteins and expressed in pichia pastoris system
- VernacularTitle:凋亡抑制因子Survivin基因的克隆及其在Pichia pastoris中的表达
- Author:
Yuehua WANG
;
Qingyun ZHANG
- Publication Type:Journal Article
- Keywords:
Survivin;
Apoptosis inhibitor;
Pichia pastoris expression;
Tumor
- From:
Cancer Research and Clinic
1999;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone human survivin gene and express survivin-an inhibitor of apoptosis proteins in Pichia pastoris eukaryotic expression system. Methods Full length survivin gene was amplified by PCR using survivin specific primers. The verified survivin gene by sequencing was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear survivin gene was introduced into GS115/ His-cells by electroporation. The transformants were transferred onto YPD plates that contained different concentrations of G418 for screening the positive clones. The integrated survivin gene in positive clones was confirmed by PCR. Selected transformants were cultured in BMMY medium with 1 % methanol for inducing the expression of survivin protein. The expressed survivin protein was analyzed by ELISA. Results The cloned human survivin sequence was the same as that in the GeneBank. The recombinant pPic9k-survivin was constructed in Picha pastoris eukaryotic expression vector. After the linear digestion, the recombinant vector was introduced into GS115/ His-cells, the 6 positive clones against G418(4 mg/mL) were obtained and confirmed by PCR; the highest survivin protein was expressed when expressed for two days in 1 % methanol BMMY medium. Conclusions Improved survivin protein yield may be reached by modifying the experimental conditions. This will help to further study the biological functions of survivin and its roles in tumor developments.
