本文關(guān)鍵詞：建立一種快速檢測NF-κB活性的酶標(biāo)方法　出處：《浙江大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人NF-kBP50基因 多克隆抗體 NF-kB活性檢測

【摘要】：NF-κB是一種十分重要的轉(zhuǎn)錄因子,參與多種基因的表達(dá)調(diào)控,具有影響細(xì)胞的生長、分化、凋亡、癌變和個(gè)體發(fā)育等多種生物學(xué)功能。它的異常激活與腫瘤的發(fā)生,抗藥性和腫瘤的轉(zhuǎn)移相關(guān),而抑制細(xì)胞內(nèi)NF-κB的異常激活就可抑制腫瘤的發(fā)生、轉(zhuǎn)移,降低腫瘤細(xì)胞對化療或放療的耐受性。因此,NF-κB是一種新型抗腫瘤藥物篩選的靶分子。 本實(shí)驗(yàn)從人神經(jīng)膠質(zhì)瘤U251細(xì)胞中提取總RNA,通過RT-PCR方法得到人NF-κB P50全長基因(長1308bp,編碼436個(gè)氨基酸);構(gòu)建了重組表達(dá)載體pGEX-4T-P50;轉(zhuǎn)入大腸桿菌后,在30℃,IPTG終濃度為0.2mM,誘導(dǎo)6hr,獲得融合蛋白GST-P50(分子量約70kD),表達(dá)量占菌體總蛋白的30%,可溶性融合蛋白為總?cè)诤系鞍椎?/5;純化融合蛋白并免疫家兔獲得了多克隆抗體,抗體經(jīng)純化并分裝保存;Western印跡分析表明純化后的多克隆抗體中已經(jīng)無針對GST蛋白的抗體,而只具有較強(qiáng)的專一于P50蛋白的抗體;ELISA分析證實(shí)其效價(jià)為5×10~5。 本實(shí)驗(yàn)構(gòu)建的酶標(biāo)檢測方法,經(jīng)過摸索并確定了:GST-P50包被濃度為1μg/ml、包被體積為100μl,抗體的稀釋度為1/1600,抗體與核蛋白樣品的溫育條件為37℃ 1hr,此時(shí)檢測NF-κB活性的靈敏度最高,可達(dá)到ng級(jí),足以檢測出細(xì)胞中NF-κB活性的微量變化。 進(jìn)一步利用檢測模型來驗(yàn)證此酶標(biāo)方法的優(yōu)劣,實(shí)驗(yàn)組加入了100ng/mlLPS刺激U251細(xì)胞30min,細(xì)胞中NF-κB的活性就顯著增高,1hr達(dá)到峰值,然后開始下降,6hr已經(jīng)處于低水平的維持狀態(tài);當(dāng)刺激時(shí)間同樣維持在1hr,LPS的濃度分別為10ng/ml、20ng/ml、40ng/ml、60ng/ml、100ng/ml時(shí),細(xì)胞中NF-κB的活性隨劑量的增加而增高,用我們的酶標(biāo)方法得到的檢測結(jié)果(細(xì)胞內(nèi)NF-κB活性變化趨勢)與Wang X的報(bào)道一致,從而驗(yàn)證了本實(shí)驗(yàn)所建立的酶標(biāo)方法具有特異性和實(shí)效性。該酶標(biāo)方法的建立為判定NF-κB的活化程度、研究相關(guān)信號(hào)轉(zhuǎn)導(dǎo)及篩選高效的NF-κB拮抗藥物奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R341

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