本文選題：DR5　切入點(diǎn)：凋亡　出處：《河南大學(xué)》2008年碩士論文

【摘要】： 背景 TRAIL(TNF-related apoptosis-inducing ligand)是屬于能夠誘導(dǎo)細(xì)胞凋亡的TNF超家族成員。作為二型跨膜蛋白,它能夠引起包括腫瘤細(xì)胞及一些轉(zhuǎn)化細(xì)胞的凋亡。而DR5是TRAIL的重要受體之一,在TRAIL誘導(dǎo)細(xì)胞凋亡中起著重要的作用。DR5或DR4特異性的抗體的作用將優(yōu)于外源性TRAIL的應(yīng)用,因?yàn)橐恍┠[瘤細(xì)胞的表面不僅有DR5/DR4,而且還有誘騙受體的表達(dá),使TRAIL的作用發(fā)揮受到限制,但是特異性的DR5抗體不會(huì)影響它的凋亡誘導(dǎo)作用。針對(duì)能夠特異的通過和死亡受體結(jié)合而向下傳遞凋亡信號(hào),引起靶細(xì)胞的凋亡的特點(diǎn),DR5抗體克服了TRAIL的不良作用,成為理想的候選者。盡管國內(nèi)外已經(jīng)制備出一些DR5的抗體,其中有一種抗體已經(jīng)進(jìn)入臨床一期的實(shí)驗(yàn)階段,但是,至今仍沒有上市的有效藥物。 目的 對(duì)已獲得的小鼠抗人DR5單克隆抗體的特異性、抗體亞型以及生物學(xué)功能進(jìn)行鑒定,并對(duì)抗體通過DR5進(jìn)一步誘導(dǎo)腫瘤細(xì)胞凋亡的機(jī)制進(jìn)行探討。 方法 將表達(dá)載體pET30a/DR5轉(zhuǎn)化到BL21,經(jīng)0.1mM IPTG誘導(dǎo)表達(dá),鎳柱純化后,經(jīng)SDS-PAGE、Western-blot和蛋白質(zhì)譜分析鑒定抗原蛋白。以表達(dá)純化的DR5抗原蛋白免疫Balb/c小鼠,經(jīng)融合、篩選及反復(fù)克隆化為基礎(chǔ)(抗體制備過程由天廣實(shí)生物技術(shù)有限公司完成),對(duì)獲得的抗人DR5單抗-WD1進(jìn)行鑒定,Western Blot和FACS方法檢測單抗WD1與重組的DR5及膜型DR5的結(jié)合。MTT法檢測WD1對(duì)幾種細(xì)胞的生長抑制作用。吉姆薩染色、Annexin V/PI雙染檢測WD1對(duì)Jurkat的凋亡作用。通過將pcDNA3.1/DR4重組質(zhì)粒轉(zhuǎn)染至293T細(xì)胞以FACS檢測WD1與DR4的交叉反應(yīng)性。通過RT-PCR的方法釣取Jurkat和Daudi細(xì)胞的DR5胞內(nèi)段蛋白序列分析細(xì)胞的突變情況,做DISC免疫沉淀實(shí)驗(yàn)分析抗體與DR5激活的關(guān)系。 結(jié)果 原核表達(dá)載體在BL21中實(shí)現(xiàn)大量表達(dá),經(jīng)SDS-PAGE和蛋白質(zhì)譜分析鑒定,在19kD處有一明顯的誘導(dǎo)表達(dá)帶,其分子量大小與預(yù)期相符。Western-blot分析表明,該表達(dá)蛋白帶可被抗人DR5抗體特異性的識(shí)別。為單抗的鑒定奠定了基礎(chǔ)。成功制備了一株活性較好的抗DR5單克隆抗體-WD1。亞型分析WD1的重鏈為IgG1型,輕鏈為κ型。WD1可識(shí)別19KD的DR5單體蛋白,而且也識(shí)別37KD的DR5二聚體蛋白。FACS分析結(jié)果提示W(wǎng)D1可與多種細(xì)胞的膜型DR5分子特異性結(jié)合,而與DR4+/293T細(xì)胞無交叉反應(yīng)。MTT結(jié)果表明WD1對(duì)Jurkat、Molt-4細(xì)胞增殖的抑制作用呈劑量依賴性;細(xì)胞形態(tài)學(xué)(吉姆薩染色)和FACS ( Annexin V/PI)分析結(jié)果證實(shí)WD1對(duì)Jurkat細(xì)胞具有誘導(dǎo)凋亡效應(yīng)。 通過RT-PCR的方法釣取Jurkat和Daudi細(xì)胞的DR5胞內(nèi)段蛋白序列,經(jīng)比對(duì)測序結(jié)果,兩種細(xì)胞均與文獻(xiàn)報(bào)道的DR5胞內(nèi)段序列完全一致,這樣就證實(shí)了細(xì)胞系本身未發(fā)生突變,不影響DR5的傳導(dǎo)功能。而DISC的免疫沉淀實(shí)驗(yàn)顯示W(wǎng)D1之所以能誘導(dǎo)Jurkat細(xì)胞的凋亡,是因?yàn)閃D1作用Jurkat細(xì)胞后,可以募集FADD和caspase-8,形成DISC復(fù)合物,繼而引起細(xì)胞凋亡的發(fā)生。 結(jié)論 (1)獲得具有生物活性的DR5胞外段蛋白。 (2)制備一株有凋亡活性的抗DR5單克隆抗體WD1。 (3)DISC復(fù)合物的形成是單克隆抗體WD1激活細(xì)胞DR5發(fā)生凋亡的原因。
[Abstract]:Background


TRAIL ( TNF - related apoptosis - inducing ligand ) is a member of the TNF superfamily that can induce apoptosis . As a two - type transmembrane protein , it can induce apoptosis including tumor cells and some transformed cells . DR5 is one of the important receptors of TRAIL and plays an important role in TRAIL - induced apoptosis .


Purpose


The specificity , antibody subtype and biological function of anti - human DR5 monoclonal antibody in mice were identified , and the mechanism of anti - apoptosis of tumor cells was further investigated by DR5 .


method


The expression vector pET30a / DR5 was transformed into BL21 , the expression was induced by 0.1 mM IPTG . After purification of nickel column , the binding of WD1 to DR5 and DR5 was determined by SDS - PAGE , Western - blot and protein analysis .


Results


Western - blot analysis showed that WD1 could bind to DR5 molecule of multiple cells . The results suggested that WD1 could bind to DR5 molecule of multiple cells without cross reaction .


By RT - PCR , the sequence of DR5 intracellular segment was obtained by RT - PCR . The results showed that both cells were identical to DR5 intracellular sequences reported in the literature , which confirmed that the cell line itself had no mutation , which did not affect the conduction function of DR5 .


Conclusion


( 1 ) obtaining a DR5 extracellular domain protein having biological activity .


( 2 ) preparing an anti - DR5 monoclonal antibody WD1 with apoptosis activity .


( 3 ) The formation of DISC complex is the cause of apoptosis of cell DR5 activated by monoclonal antibody WD1 .

【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R730.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李淑蓮;張軍;劉廣超;白慧玲;劉英杰;盧峰;馬遠(yuǎn)方;;抗人DR5單克隆抗體(mDRA-6)誘導(dǎo)Jurkat細(xì)胞凋亡活性研究[J];中國免疫學(xué)雜志;2006年01期

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本文編號(hào)：1678973