摘要
目的:构建人肿瘤抑素(tumstatin)活性片段Tum5的融合重组表达载体并对其进行诱导表达和蛋白纯化以获得大量重组融合蛋白.方法:人工合成Tum5基因,进行测序分析,将该基因克隆入原核表达载体pQE30中,转化感受态细胞DH5α,经IPTG诱导表达重组融合蛋白,对表达产物进行TricineSDSPAGE电泳分析和WesternBlot检测分析.结果构建了重组融合表达质粒pQE30Tum5,表达的蛋白经TricineSDSPAGE电泳分析,在约Mr10×103处出现了一条新生的蛋白条带,经灰度扫描检测,表达量约占菌体总蛋白的40%,纯化后的蛋白灰度扫描检测,纯度为95%.结论:成功地构建了人Tum5基因的原核表达载体,并纯化了该基因的原核表达产物.
AIM: To construct the recombinant expression vector of Tum-5 and to purify and identify the fusion protein rhTum-5. METHODS: A 264 bp of human Tum-5 gene fragment was synthesized and cloned into pQE-30 vector, an E.coli expression vector. The plasmid was transformed into E.coli DH5α and induced to express fusion protein rhTum-5 with IPTG. The expression of Tum-5 was detected by Tricine-SDS-PAGE and Western blot. RESULTS: A novel protein with expected molecular mass was expressed upon induction with IPTG. The expressed product accounted for about 40% of total bacterial proteins and showed good reactivity to anti-His tag antibody. CONCLUSION: Our successful cloning and expression of human Tum-5 gene and purification of rhTum-5 protein lay a basis for further study on the application of this protein to anti-angiogenesis in vitro and in vivo.
出处
《第四军医大学学报》
北大核心
2005年第14期1279-1281,共3页
Journal of the Fourth Military Medical University
