基因重组TNFα衍生物TRSP10的高效制备及其对DU145细胞抑制作用研究

Efficient Preparation of TNFα Derivatives TRSP10 and Preliminary Study of Its Inhibitory Effect on Prostate Cancer DU145 Cells

利用基因工程技术表达能够促使肿瘤细胞DU145凋亡的肿瘤坏死因子α(TNFα)的衍生物TRSP10,并在体外研究其对DU145细胞的抑制效应。以重叠延伸PCR方法合成TRSP10基因序列,并插入高效表达的质粒载体p KYB-MCS的NdeⅠ和SapⅠ酶切位点之间,优化融合蛋白诱导表达的条件,建立了从载体构建到重组菌表达、制备的工艺技术条件。MTT法检测TRSP10对前列腺癌细胞DU145增殖的抑制作用。实验结果表明:重组菌ER2566诱导表达可溶性融合蛋白的最佳条件是诱导剂IPTG浓度为0.8 mmol/L、诱导表达温度37℃、诱导表达时间8h。利用IMPACT系统及HPLC技术纯化制备TRSP10,得到产物纯度达到96%,质谱鉴定确定其分子质量为3.59k Da,与理论值相符;体外细胞学研究结果表明,TRSP10对前列腺癌细胞DU145有明显的抑制作用,在5,10,20,40μmol/L TRSP10及10μmol/L TNFα阳性对照处理后48h抑制率分别达到11.40%,22.97%,33.26%,48.35%及42.50%。

It is intending to prepare tumor necrosis factor α （TNFα） derivative TRSP10 which can lead prostate cancer DU145 cells apoptosis by genetic engineering and to study the inhibitory effect on DU145 cells in vitro. Trsp 10 gene sequences were synthesized by overlap extension PCR and then inserted into the site between SapI and NdeI in the highly-efficient expression vector,pKYB-MCS and conduct the optimal conditions to induce the expression of the fusion protein and set up the technology from vector construction to the expression and purification of the recombinant bacteria. MTT detect the inhibit proliferation effect of the TRSP10 on prostate cancer DU145 cells. The results showed that the optimal expression condition was as follows： at 0.8 mmol /L IPTG,37 ℃ and 8 hours.Puried by the IMPACT system and HPLC technology, the purity of the TRSP10 is 96%.The molecular mass is 3.59kDa that is determined by the mass spectrometry, which is consistent with the theoretical data. In vitro, studies showed that, TRSP10 could significantly inhibit the proliferation of prostate cancer DU145 cells.The rates of inhibition among the concentrations of 5,10,20,40μmol/L, are 11.40%, 22.97%, 33.26%, 48.35% respectively.