人肿瘤抑素(tumstatin)的基因克隆及其原核表达

Cloning and expression of human tumstatin gene in E.coli

目的克隆人肿瘤抑素(tumstatin)基因,并进行其在大肠杆菌表达的研究。方法用RT-PCR技术从人肾癌旁组织中扩增出肿瘤抑素的cDNA,构建原核表达载体pQE-tum,IPTG诱导重组蛋白质的表达,并利用Ni-NTAHis-Bind树脂纯化该重组蛋白质。结果经PCR扩增成功获得742 bp的人肿瘤抑素基因,测序正确。在大肠杆菌中目的蛋白质的表达量占菌体总蛋白质的10%,SDS-PAGE及Western blot分析显示,其相对分子质量为28 000,纯化后的6×His-tumstatin纯度可达92%。结论tumstatin基因克隆、表达及纯化的成功,为其今后的肿瘤抗血管生成治疗研究奠定了基础。

Purpose To clone and express recombinant human tumstatin in E. coli. Methods The human tumstain gene was isolated with RT-PCR from human renal tissue total RNA and cloned into PGEM-T vector for sequencing. Then,the tumstatin gene was subcloned into the pQE-30 expression vector and was transformed into E. coli. The recombinant tumstatin was purified with Ni-NTA His-Bind resin. Results The DNA sequence of cloned tumstatin gene was similar to that reported previously. The construct was expressed in E. coli with high level, accounting for 10% of the total bacterial proteins. This gene product was found with a molecular mass of 28000 on SDS-PAGE and Western blot, which was identical to that of native tumstatin. The purity of recombinant tumstatin purified by affinity chromatography reached more than 92%. Conclusion The cloning, expression and purification of human tumstatin has laid a foundation for its antiangiogenesis theraphy of tumor.