Persephin基因克隆及在大肠杆菌中的表达

目的 :获取Persephin(PSP)基因并实现其高效表达。 方法 :利用PCR方法以染色体DNA为模板 ,扩增编码人PSP成熟蛋白的基因片段 ,将其克隆于 pUC19质粒 ,进行序列分析 ,将基因重组于硫氧还蛋白融合表达载体pThioHisA并进行表达。结果 :序列分析结果与文献报道一致 ,表达的hPSP占菌体总蛋白质 15 %左右。结论 :人PSP基因在大肠杆菌Top10中获得了高效、稳定表达 ,为进一步的基础研究与临床应用奠定了基础。

Cloning of Human Persephin Gene and Its Expression in E.coli

Purpose:To obtain cDNA and high level expression in E.coli of human Persephin(PSP). Methods:The cDNA fragment encoding mature hPSP was amplified by PCR from human chromosome DNA extracted from liver tissue, and ligated into cloning vector pUC19. The nucleotides sequence was identified by Sanger method. The gene was inserted into thioredoxin fusion expression vector pThioHis A between EcoR Ⅰ and Xbal Ⅰ sites. The recombinant plasmid was transformed into E.coli strain Top10 and induced with 1 mmol/L IPTG.Results:Sequence analysis verified that the fragment cloned in pUC19 was hPSP cDNA. Higher levels of expression of hPSP thioredoxin fusion protein was obtained at 6 or 8 hours after induction. The expressed fusion protein, with molecular weight of about 27kD, is about 15% of the total bacteria protein as detected by SDS PAGE and densitometry analysis.Conclusion:This work provided us a basis for the further basic study and clinical application of hPSP.