人胃癌组织中期因子的克隆表达及其对NIH3T3细胞生长促进作用

根据GenBank报道的序列,设计一对引物,通过RT-PCR从胃肿瘤患者肿瘤组织中获得了Midkine(MK)成熟肽DNA编码序列,与pMD18T-vector连接测序后,将该片段克隆入大肠杆菌表达载体pET30(a+)中,转化大肠杆菌表达菌株BL21(DE3),筛选得到可诱导表达MK重组蛋白的工程菌株pEMK,经IPTG低温诱导表达产生的总可溶蛋白用Heparin结合的亲和柱纯化,并用MTT法验证表明大肠杆菌表达产生的可溶性MK蛋白具有促进NIH3T3细胞增殖的作用。

Cloning and expression of Midkine cytokine from carcinogenesis tissue in E.coli

For cloning the cytokine human Midkine (MK) gene, we designed a pair of PCR specific primers according to the reported human MK cDNA sequence in GenBank. The target DNA fragment was obtained by RT-PCR from gastric carcinoma patient's carcinogenesis tissue and cloned into pMD18 T-vector. After sequencing the MK nucleotide fragment was inserted into an E.coli expression vector pET30(a+).The recombinant plasmid (30 MK) was obtained and transferred into E.coli BL21 (DE3), the resulted E.coli pEMK was cultured and induced with IPTG, the efficiently expressed recombinant MK protein was purified from the total cellular soluble protein by Heparin Sepharose 4B column. Moreover, MTT methods was used and determined that the recombinant MK protein possessed the activity to promote the growth of NIH3T3 cells.