重组人生存素单体分子与双体分子的原核表达研究

Prokaryotic expression of recombination human single and double molecular survivin

目的重组并高效表达人生存素单体分子和双体分子。方法PCR扩增人生存素基因,将其单一读码框和双读码框重组到原核表达质粒载体pKpL5的表达框内,限制性内切酶分析后,于42℃诱导表达,并以SDS-PAGE和Western blotting鉴定表达结果。结果经限制性核酸内切酶分析,表达重组人生存素单体分子质粒pKpL5-sSURVIVIN和表达人生存素同源双体分子质粒pKpL5-dSURVIVIN均正确构建,可高效表达分子量约18.5KDa的人生存素单体分子与分子量约35KDa的同源双体分子,所表达的目的蛋白可被抗人生存素的多克隆抗体特异性识别。结论该研究成功高效表达人生存素单体分子与同源双体分子,为进一步研究其生物活性与免疫原性奠定了物质基础。

[Objective] To construct and express the human single and double Molecular survivin in prokaryotic cell. [Methods] Survivin cDNA amplified with PCR, and the single and double open reading frame of survivin was inserted into pKpL-5 respectively. After identified with restriction endonuclerase, the two recombinant plasmids were transformed into E. coli pep2136 and induced at 42~C for 4 hours, the expressed protein was analyied by SDS- PAGE and Western blotting. [Results] The two recombinant plasmids were constructed successfully, and the protein were expressed effectively, and the relative molecular weight were 18.5 KDa, 35 KDa respectively. Western-blot analysis demonstrated that the expressed protein exhibited specific reaction with rabbit anti-survivin pelyclone anti- body. [Conclusion] The single and double molecular survivin was expressed effectively. It lays foundation .for further study the antigenicity and the biological activity.