摘要
利用基因重组技术制备具有抗肿瘤作用的肿瘤坏死因子-α(TNF-α)衍生多肽RMPT,并研究其体外生物学效应。PCR方法合成RMPT基因序列,定向插入高效表达载体pTXB1-MCS,在大肠杆菌ER2566中诱导表达重组融合蛋白并建立RMPT融合蛋白诱导表达的优化体系。N端自动切割、纯化、HPLC制备获得RMPT,电泳和质谱鉴定其分子质量。CCK-8检测RMPT对肿瘤细胞的抑制作用。实验结果表明:重组工程菌可溶性RMPT融合蛋白最佳诱导表达条件为37℃、8 h、IPTG浓度0.75 mmol/L;DTT诱导蛋白内含肽自剪切固相纯化和HPLC制备重组肽RMPT,纯度大于95%,电泳和质谱鉴定其分子质量为3.7 ku,结果与理论值相符合,其产率为10.8 mg/L发酵液;体外研究表明,RMPT能够高效抑制多数肿瘤细胞增殖。
This experiment is intending to prepare the tumor necrosis factor-or （TNF-α） derivate peptides RMPT which haveantitumor function by gene recombination technique and to study its biological effects in vitro. RMPT gene sequences were synthesized by PCR and then inserted directionally into highly-efficient expression vector, pTXB1-MCS. The recombined fusion protein was induced and expressed in E. coli ER2566 and the optimal system which was induced and expressed by the RMPT fusion protein was set up. The RMPT fusion protein could be obtained by purification, automatic cut of N-terminus and HPLC. And its molecular weight could be identified by means of electrophoresis and mass spectra. CCK-8 could detect the RMPT＇ s ability to inhibit cancer cells growth. The results showed that the optimal expression condition of the recombined engineering bacteria and soluble RMPT fusion protein was as follows ： at 37 ℃, 8 hours and 0.75 mmol/L IPTG. Resorting to HPLC as well as DT＇F induction of self-splicing and solid phase purification of intein in proteins , the recombined peptides, RMPT, could be prepared with over 95 % purity. The results of electrophoresis and mass spectra accorded with the theoretical data and its yield was 10.8 mg/L fermentation product. In vitro, studies showed that the RMPT could significantly inhibit the cancer cells proliferation with a high rate of inhibitation.
出处
《药物生物技术》
CAS
2013年第1期21-25,共5页
Pharmaceutical Biotechnology
基金
广东省自然科学基金项目(No.S2012010008756)
高等学校博士学科点专项科研基金项目(20124401120012)
广东省高等学校科技创新项目(2012KJCX0015)
广东省教育部产学研结合项目(No.2010B090400544)
广州市科技计划项目(No.2011J4300112)
中央高校基本科研业务费专项资金资助项目(No.21612408)
科技部科技型中小企业技术创新基金项目(No.11C26214413218)
广州市科技计划项目(No.2010Z2-C321)
国家"863"项目(No.2006AA02Z125)
关键词
TNF-α衍生多肽
基因重组
表达优化
抗肿瘤作用
TNF-α derivate peptide, Genetic recombination, Expression optimization, Antitumor effects
