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

doi:10.13523/j.cb.20150402

中国生物工程杂志

2015, Vol. 35

Issue (4): 11-16 DOI: 10.13523/j.cb.20150402

研究报告

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

方世雄, 马义, 沈淑桃, 赵绍军, 洪岸

暨南大学细胞生物学系暨南大学生物医药研究院广东省生物工程药物重点实验室基因工程药物国家工程研究中心广州 510632

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

FANG Shi-xiong, MA Yi, SHEN Shu-tao, ZHAO Shao-jun, HONG An

Department of Cell Biology of Jinan University, Institute of Biological Medicine of Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China

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摘要：

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pKYB-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3.59kDa,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%。

关键词： 基因工程; 肿瘤坏死因子α 衍生物; 表达纯化; 抑制增殖

Abstract:

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.

Key words: Genetic engineering Tumor necrosis factor alpha derivative Expression and purification Inhibit proliferation

收稿日期: 2015-01-04 出版日期: 2015-04-25

ZTFLH:

Q789

基金资助:

国家自然科学基金面上项目(81373314)、广东省自然科学基金(S2012010008756)、高等学校博士学科点专项科研基金项目(20124401120012)、中央高校基本科研业务费专项资金(21612408)、科技部科技型中小企业技术创新基金(11C26214413218)、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(2010B090400544,2013B090500105)、广州市科技计划项目(2011J4300112)资助项目

通讯作者: 马义 E-mail: tmayi@jnu.edu.cn

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引用本文:

方世雄, 马义, 沈淑桃, 赵绍军, 洪岸. 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[J]. 中国生物工程杂志, 2015, 35(4): 11-16.

FANG Shi-xiong, MA Yi, SHEN Shu-tao, ZHAO Shao-jun, HONG An. Efficient Preparation of TNFα Derivatives TRSP10 and Preliminary Study of Its Inhibitory Effect on Prostate Cancer DU145 Cells. China Biotechnology, 2015, 35(4): 11-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150402 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I4/11

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