發(fā)布時(shí)間：2018-03-13 08:31

  本文選題：hPARP1　切入點(diǎn)：PARP抑制劑　出處：《江南大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：聚ADP核糖聚合酶（Poly（ADP-ribose）polymerase，PARP）是一種參與DNA修復(fù)的核酶，在DNA損傷修復(fù)中起重要作用，為腫瘤的治療提供一個(gè)新的靶點(diǎn)。因此，PARP抑制劑一直是近年來的研究熱點(diǎn)，部分抑制劑已進(jìn)入臨床試驗(yàn)階段。本論文對(duì)PARP抑制劑進(jìn)行體外篩選并初步評(píng)價(jià)其藥效，這為篩選有自主知識(shí)產(chǎn)權(quán)的新型PARP抑制劑奠定了基礎(chǔ)。 本課題通過基因重組技術(shù)將hPARP1基因插入到pFastBacTM1載體中，獲得質(zhì)粒pFast-hPARP1；將pFast-hPARP1轉(zhuǎn)化入DH10Bac感受態(tài)細(xì)胞中，通過位點(diǎn)特異性轉(zhuǎn)座，hPARP1基因可以整合入Bacmid穿梭載體中，獲得表達(dá)質(zhì)粒Bacmid-hPARP1；轉(zhuǎn)染Bacmid-hPARP1至Sf9昆蟲細(xì)胞進(jìn)行表達(dá)，表達(dá)產(chǎn)物經(jīng)3-氨基苯甲酰胺親和層析柱分離純化并通過SDS-PAGE、Western blotting和酶活檢測(cè)法進(jìn)行鑒定。通過測(cè)定已知PARP抑制劑DPQ和菲啶酮PHE對(duì)昆蟲表達(dá)hPARP1的IC_(50)，驗(yàn)證此方法可以用來進(jìn)行新型PARP抑制劑的篩選。通過克隆形成實(shí)驗(yàn)和CCK8細(xì)胞增殖-毒性檢測(cè)實(shí)驗(yàn)，篩選對(duì)PARP抑制劑PHE敏感的細(xì)胞，用于篩選PARP抑制劑的細(xì)胞模型。 實(shí)驗(yàn)結(jié)果表明，本論文成功構(gòu)建了pFast-hPARP1質(zhì)粒，通過Bac-to-Bac桿狀病毒表達(dá)系統(tǒng)，實(shí)現(xiàn)了hPARP1酶在Sf9昆蟲細(xì)胞中的表達(dá)，產(chǎn)物經(jīng)3-氨基苯甲酰胺柱親和層析成功得到純化，得率為80.8%，純化倍數(shù)為38.72倍，比活達(dá)到1.988U/μg。SDS-PAGE和Western blotting結(jié)果顯示純化產(chǎn)物在116kDa處有特異性目標(biāo)條帶。PARP抑制劑體外篩選結(jié)果顯示，在72種化合物中篩選出41個(gè)具有生物活性的PARP抑制劑，其中有7個(gè)化合物的IC_(50)低于500nmol/L，顯示出良好的PARP抑制作用。細(xì)胞模型篩選結(jié)果顯示，中國人源胰腺癌細(xì)胞JF305對(duì)PARP抑制劑菲啶酮具有一定的敏感性。CCK8結(jié)果顯示，篩選獲得的PARP抑制劑HC-232（A）具有明顯抑制JF305細(xì)胞的生長(zhǎng)作用。 本課題通過分子構(gòu)建、表達(dá)、純化等步驟獲得了hPARP1。利用hPARP1，成功篩選出具有一定生物活性的PARP抑制劑。獲得了對(duì)PARP抑制劑菲啶酮敏感的中國人源胰腺癌細(xì)胞JF305，這為篩選出具有自主知識(shí)產(chǎn)權(quán)的PARP抑制劑奠定了基礎(chǔ)，并對(duì)后續(xù)研究提供了理論依據(jù)。
[Abstract]:Poly (ADP) ribosome polymerase (ADP) is a ribozyme involved in DNA repair, which plays an important role in the repair of DNA and provides a new target for tumor treatment. Some of the inhibitors have entered the stage of clinical trial. In this paper, PARP inhibitors were screened in vitro and their efficacy was preliminarily evaluated, which laid a foundation for the screening of new PARP inhibitors with independent intellectual property rights. In this study, the hPARP1 gene was inserted into the pFastBacTM1 vector by gene recombination technique, and the plasmid pFast-hPARP1 was obtained, and the pFast-hPARP1 was transformed into the DH10Bac receptive cells, which could be integrated into the Bacmid shuttle vector by site-specific transposition of hPARP1 gene. The expression plasmid Bacmid-hPARP1 was obtained and transfected into Sf9 insect cells for expression. The expressed product was separated and purified by 3- aminobenzoamide affinity chromatography and identified by SDS-PAGEG Western blotting and enzyme activity assay. It was proved that this method could be used to detect the expression of hPARP1 in insects by detecting the known PARP inhibitors DPQ and phenidinone PHE. Screening of novel PARP inhibitors. Clone formation assay and CCK8 cell proliferation-toxicity test were used. Screening of cells sensitive to PARP inhibitor PHE was used to screen cell models of PARP inhibitors. The results showed that the pFast-hPARP1 plasmid was successfully constructed and the expression of hPARP1 in Sf9 insect cells was realized by Bac-to-Bac baculovirus expression system. The product was purified by 3- aminobenzoamide column affinity chromatography. The specific activity of the purified product was 1.988 U / 渭 g. SDS-PAGE and Western blotting. The results showed that the purified product had a specific target band at 116 kDa. The in vitro screening results showed that 41 bioactive PARP inhibitors were screened out of 72 compounds. In 7 of them, the ICS 50) was lower than 500nmol / L, showing a good inhibitory effect on PARP. The cell model screening showed that JF305, a Chinese pancreatic cancer cell line, was sensitive to the PARP inhibitor phenidronone. CCK8 showed that, The selected PARP inhibitor, HC-232, can significantly inhibit the growth of JF305 cells. This topic is expressed by molecular construction, PARP inhibitors with certain biological activity were successfully screened by using hPARP1.The Chinese pancreatic cancer cell line JF305, which was sensitive to PARP inhibitor phenidinone, was obtained, which was used to screen PARP with independent intellectual property rights. The inhibitor laid the foundation, It also provides the theoretical basis for the follow-up study.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：TQ464.8

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本文編號(hào)：1605598