建立一种快速检测NF-κB活性的酶标方法

发布时间：2018-01-07 08:11

本文关键词：建立一种快速检测NF-κB活性的酶标方法　出处：《浙江大学》2005年硕士论文　论文类型：学位论文

【摘要】：NF-κB是一种十分重要的转录因子,参与多种基因的表达调控,具有影响细胞的生长、分化、凋亡、癌变和个体发育等多种生物学功能。它的异常激活与肿瘤的发生,抗药性和肿瘤的转移相关,而抑制细胞内NF-κB的异常激活就可抑制肿瘤的发生、转移,降低肿瘤细胞对化疗或放疗的耐受性。因此,NF-κB是一种新型抗肿瘤药物筛选的靶分子。本实验从人神经胶质瘤U251细胞中提取总RNA,通过RT-PCR方法得到人NF-κB P50全长基因(长1308bp,编码436个氨基酸);构建了重组表达载体pGEX-4T-P50;转入大肠杆菌后,在30℃,IPTG终浓度为0.2mM,诱导6hr,获得融合蛋白GST-P50(分子量约70kD),表达量占菌体总蛋白的30%,可溶性融合蛋白为总融合蛋白的1/5;纯化融合蛋白并免疫家兔获得了多克隆抗体,抗体经纯化并分装保存;Western印迹分析表明纯化后的多克隆抗体中已经无针对GST蛋白的抗体,而只具有较强的专一于P50蛋白的抗体;ELISA分析证实其效价为5×10~5。本实验构建的酶标检测方法,经过摸索并确定了:GST-P50包被浓度为1μg/ml、包被体积为100μl,抗体的稀释度为1/1600,抗体与核蛋白样品的温育条件为37℃ 1hr,此时检测NF-κB活性的灵敏度最高,可达到ng级,足以检测出细胞中NF-κB活性的微量变化。进一步利用检测模型来验证此酶标方法的优劣,实验组加入了100ng/mlLPS刺激U251细胞30min,细胞中NF-κB的活性就显著增高,1hr达到峰值,然后开始下降,6hr已经处于低水平的维持状态;当刺激时间同样维持在1hr,LPS的浓度分别为10ng/ml、20ng/ml、40ng/ml、60ng/ml、100ng/ml时,细胞中NF-κB的活性随剂量的增加而增高,用我们的酶标方法得到的检测结果(细胞内NF-κB活性变化趋势)与Wang X的报道一致,从而验证了本实验所建立的酶标方法具有特异性和实效性。该酶标方法的建立为判定NF-κB的活化程度、研究相关信号转导及筛选高效的NF-κB拮抗药物奠定了基础。
[Abstract]:NF- B is an important transcription factor, regulating the expression of genes involved in a variety of, has affected cell growth, differentiation, apoptosis, cancer and ontogeny and other biological functions. The abnormal activation and tumor metastasis it, drug resistance and tumor related, and inhibition of NF- K B anomaly activation does occur, which could inhibit tumor metastasis, decrease the tolerance of tumor cells to chemotherapy or radiotherapy. Therefore, NF- K B is a screening of new anticancer drug target molecules.
The total RNA was extracted from human glioma U251 cells, obtained the full-length human NF- kappa B P50 gene by RT-PCR method (1308bp in length, encoding 436 amino acids); recombinant expression vector pGEX-4T-P50 was constructed and transformed into E.coli; after 30 degrees, IPTG final concentration of 0.2mM, induced by 6hr, the fusion protein GST-P50 (molecular weight 70kD), the expression of the total cell protein 30%, soluble fusion protein to total fusion protein 1/5; fusion protein was purified and immunized rabbit polyclonal antibodies were obtained after purification, antibody packaging and preservation; Western blot analysis table has no antibodies against GST protein polyclonal antibody purified in the Ming Dynasty, but only has strong specific antibody to P50 protein; ELISA analysis confirmed that the titer of 5 * 10~5.
Detection method of the constructed ELISA, after groping and determined the GST-P50 concentration for coating was 1 g/ml, was volume 100 L, antibody dilution 1/1600, antibody and nuclear protein sample incubation conditions of 37 DEG 1hr, the sensitivity of detection of NF- kappa B activity can be the highest to achieve the ng level, and can detect the change of trace NF- kappa B activity in cells.
The further use of detection models to verify the calibration of this enzyme of the experimental group with 100ng/mlLPS stimulation of U251 cells 30min cells NF- kappa B activity was significantly increased, 1hr reached the peak, and then began to decline, 6hr has maintained at a low level state; when the stimulus was maintained at the same time 1hr, LPS concentration respectively. 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml, 100ng/ml, NF- in the cells of kappa B activity increased with the increase of dose, the detection results by our ELISA obtained (kappa B activity changes of NF- cells and Wang X trend) reports, which verified the established ELISA method has the specificity and effectiveness. The establishment of standard methods of the enzyme activation degree NF- kappa B, laid the foundation for the study of signal transduction and screening of efficient NF- kappa B antagonists.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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