Cao Shanshan, Liu Yan, Li Xiaohua, Zhang Yingqi, Wang Jun, Du Wenqi, Han Yu, Jin Haifeng, Zhao Lina, Wu Kaichun, Fan Daiming
State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, People's Republic of China.
Mol Biotechnol. 2009 Sep;43(1):1-7. doi: 10.1007/s12033-009-9170-z. Epub 2009 Apr 9.
A phage-displayed peptide CGNSNPKSC (GX1) was obtained previously in our lab, which could specifically bind to the vasculature of human gastric cancer. GX1-rmhTNFalpha was a fusion protein of GX1 and recombinant mutant human tumor necrosis factor alpha (rmhTNFalpha), which was designed by us with the expectation of enhancing selectivity of rmhTNFalpha. The DNA fragment encoding GX1 was cloned into the vector pBV220 with rmhTNFalpha between the EcoRI site and the BamHI site, and then expressed in Escherichia coli DH5alpha by temperature induction. Subsequently, E. coli DH5alpha was lysed, and the GX1-rmhTNFalpha protein was found in both soluble form and inclusion bodies. The protein was fractionated with ammonium sulfate deposition from 30% to 60%, and purified by cation and anion exchange chromatography using SP Sepharose Fast Flow column and Q Sepharose Fast Flow column. The purity of protein was then identified by SDS-PAGE and HPLC. Subsequent studies showed that GX1-rmhTNFalpha had high bioactivity of 5.65 x 10(8) IU/ml, which was similar with natural human TNFalpha and could reach the tumor site relatively faster than rmhTNFalpha.
我们实验室先前获得了一种噬菌体展示肽CGNSNPKSC（GX1），它能够特异性结合人胃癌的脉管系统。GX1-rmhTNFalpha是GX1与重组突变型人肿瘤坏死因子α（rmhTNFalpha）的融合蛋白，我们设计它是期望提高rmhTNFalpha的选择性。将编码GX1的DNA片段克隆到载体pBV220中，使rmhTNFalpha位于EcoRI位点和BamHI位点之间，然后通过温度诱导在大肠杆菌DH5alpha中表达。随后，裂解大肠杆菌DH5alpha，发现GX1-rmhTNFalpha蛋白以可溶性形式和包涵体形式存在。用30%至60%的硫酸铵沉淀对该蛋白进行分级分离，并使用SP Sepharose Fast Flow柱和Q Sepharose Fast Flow柱通过阳离子和阴离子交换色谱法进行纯化。然后通过SDS-PAGE和HPLC鉴定蛋白的纯度。后续研究表明，GX1-rmhTNFalpha具有5.65×10（8）IU/ml的高生物活性，这与天然人TNFalpha相似，并且比rmhTNFalpha能相对更快地到达肿瘤部位。
