人DR5胞外段基因的克
發(fā)布時間：2018-07-27 11:14
【摘要】： 背景 死亡受體5(death receptor , DR5)是腫瘤壞死因子相關(guān)的凋亡誘導配體(tumor necrosis factor related apoptosis inducing ligand, TRAIL)的主要受體之一,DR5與TRAIL結(jié)合后可以通過其胞內(nèi)的死亡結(jié)構(gòu)域傳遞凋亡信號,從而引起細胞凋亡的發(fā)生,是激活凋亡途徑的很好的靶標。TRAIL可引起許多腫瘤細胞系的凋亡,對絕大多數(shù)正常細胞不具有凋亡效應(yīng),被認為是癌癥治療的潛在的生物制劑。隨著研究的深入,出現(xiàn)了關(guān)于TRAIL可以引起正常肝臟細胞的凋亡的報道。于是人們轉(zhuǎn)入了對抗DR5單克隆抗體的研究上來。由于TRAIL的功能發(fā)揮主要靠DR5介導凋亡信號,如能得到?jīng)]有肝臟細胞毒性又能活化DR5的單克隆抗體,將可以做為TRAIL的替代物進行藥物研制。 目的 獲得具有生物活性的人DR5胞外段蛋白,制備具有活性的抗DR5單克隆抗體。 方法 通過RT-PCR從Jurkat細胞中釣取DR5的胞外段基因(55-183位氨基酸),克隆到pGEM?-T Easy載體中,經(jīng)核酸序列測定正確后,將其再次克隆入原核表達載體pET-30a中,在E.coli BL21 (DE3)實現(xiàn)蛋白表達。通過SDS-PAGE和Western blot鑒定表達產(chǎn)物。ELISA法分析其與抗DR5單克隆抗體(mAb)結(jié)合能力,用DR5胞外段競爭阻斷TRAIL對Jurkat細胞生長的抑制。通過雜交瘤技術(shù)篩選抗DR5單克隆抗體(A6)。MTT法檢測了A6對Jurkat細胞的生長抑制作用。吉姆薩染色、PI/Annexinv雙染檢測了A6對Jurkat的凋亡作用。通過競爭ELISA方法分析了A6和TRAIL的作用位點的異同。 結(jié)果 克隆到人DR5基因的胞外段基因,測序結(jié)果和報道的一致;構(gòu)建了人DR5胞外段基因的原核表達載體并實現(xiàn)在E.coli BL21 (DE3)中大量表達;SDS-PAGE表明所表達蛋白與預(yù)期蛋白分子量一致;Western blot及ELISA的結(jié)果表明該蛋白能與抗DR5抗體反應(yīng),競爭阻斷試驗表明DR5胞外段可阻斷TRAIL對Jurkat細胞生長的抑制。 成功制備了一株活性較好的抗DR5單克隆抗體-A6。MTT法顯示了A6對Jurkat細胞的生長抑制作用,在25μg/mL的濃度下,作用16小時,可以達到對Jurkat細胞90㳠的生長抑制率。吉姆薩染色從形態(tài)上顯示了Jurkat細胞的凋亡。PI/AnnexinⅤ雙染分析顯示:Jurkat細胞經(jīng)過250 ng/mL濃度的A6作用12小時后,Jurkat細胞的凋亡率可以達到52.46㳠。用40倍濃度的TRAIL和A6做競爭ELISA,結(jié)果顯示了TRAIL和A6的作用位點是不同的。 結(jié)論 (1)成功地制備了具有生物活性的DR5胞外區(qū)。 (2)制備了一株抗DR5單克隆抗體A6。
[Abstract]:Background death receptor 5 (death receptor, DR5) is one of the major receptors of tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor related apoptosis inducing ligand, TRAIL), which binds to TRAIL through its intracellular Death domain transmits apoptosis signal. It is a good target of activating apoptotic pathway. Trail can induce apoptosis of many tumor cell lines and has no apoptotic effect on most normal cells. It is considered to be a potential biological agent for cancer therapy. With the development of research, there have been reports that TRAIL can induce apoptosis of normal liver cells. So people turned to the study of monoclonal antibodies against DR5. Because the function of TRAIL is mainly mediated by DR5, if we can get monoclonal antibody which can activate DR5 without hepatocytotoxicity, it can be used as a substitute for TRAIL. Objective to obtain a bioactive human DR5 extracellular segment protein and prepare anti-DR5 monoclonal antibody. Methods the extracellular segment gene (55-183 amino acids) of DR5 was isolated from Jurkat cells by RT-PCR and cloned into pGEM?-T Easy vector. After nucleic acid sequencing, the gene was cloned into prokaryotic expression vector pET-30a again. Protein expression was realized in E.coli BL21 (DE3). SDS-PAGE and Western blot were used to identify the expression product. Elisa was used to analyze the binding ability of TRAIL to (mAb), and the extracellular segments of DR5 were used to block the inhibitory effect of TRAIL on the growth of Jurkat cells. DR5 monoclonal antibody (A6). MTT assay was used to detect the inhibitory effect of A6 on the growth of Jurkat cells. The apoptotic effect of A6 on Jurkat was detected by Pi / Annexinv double staining. The similarities and differences of interaction sites between A6 and TRAIL were analyzed by competitive ELISA method. Results the extracellular gene of human DR5 gene was cloned and sequenced. The prokaryotic expression vector of human DR5 extracellular segment gene was constructed and expressed in E.coli BL21 (DE3). SDS-PAGE showed that the expressed protein had the same molecular weight as the expected protein. Western blot and ELISA showed that the protein could react with anti-DR5 antibody. Competitive blockade test showed that the extracellular segment of DR5 could block the growth inhibition of Jurkat cells induced by TRAIL. A highly active monoclonal antibody against DR5 was successfully prepared. The inhibitory effect of A6 on the growth of Jurkat cells was demonstrated by A6 method. At the concentration of 25 渭 g/mL for 16 hours, the growth inhibition of A6 on Jurkat cells reached 90%. Growth inhibition rate. Gimsa staining showed apoptosis of Jurkat cells. Pi / Annexin 鈪，
本文編號：2147641
[Abstract]:Background death receptor 5 (death receptor, DR5) is one of the major receptors of tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor related apoptosis inducing ligand, TRAIL), which binds to TRAIL through its intracellular Death domain transmits apoptosis signal. It is a good target of activating apoptotic pathway. Trail can induce apoptosis of many tumor cell lines and has no apoptotic effect on most normal cells. It is considered to be a potential biological agent for cancer therapy. With the development of research, there have been reports that TRAIL can induce apoptosis of normal liver cells. So people turned to the study of monoclonal antibodies against DR5. Because the function of TRAIL is mainly mediated by DR5, if we can get monoclonal antibody which can activate DR5 without hepatocytotoxicity, it can be used as a substitute for TRAIL. Objective to obtain a bioactive human DR5 extracellular segment protein and prepare anti-DR5 monoclonal antibody. Methods the extracellular segment gene (55-183 amino acids) of DR5 was isolated from Jurkat cells by RT-PCR and cloned into pGEM?-T Easy vector. After nucleic acid sequencing, the gene was cloned into prokaryotic expression vector pET-30a again. Protein expression was realized in E.coli BL21 (DE3). SDS-PAGE and Western blot were used to identify the expression product. Elisa was used to analyze the binding ability of TRAIL to (mAb), and the extracellular segments of DR5 were used to block the inhibitory effect of TRAIL on the growth of Jurkat cells. DR5 monoclonal antibody (A6). MTT assay was used to detect the inhibitory effect of A6 on the growth of Jurkat cells. The apoptotic effect of A6 on Jurkat was detected by Pi / Annexinv double staining. The similarities and differences of interaction sites between A6 and TRAIL were analyzed by competitive ELISA method. Results the extracellular gene of human DR5 gene was cloned and sequenced. The prokaryotic expression vector of human DR5 extracellular segment gene was constructed and expressed in E.coli BL21 (DE3). SDS-PAGE showed that the expressed protein had the same molecular weight as the expected protein. Western blot and ELISA showed that the protein could react with anti-DR5 antibody. Competitive blockade test showed that the extracellular segment of DR5 could block the growth inhibition of Jurkat cells induced by TRAIL. A highly active monoclonal antibody against DR5 was successfully prepared. The inhibitory effect of A6 on the growth of Jurkat cells was demonstrated by A6 method. At the concentration of 25 渭 g/mL for 16 hours, the growth inhibition of A6 on Jurkat cells reached 90%. Growth inhibition rate. Gimsa staining showed apoptosis of Jurkat cells. Pi / Annexin 鈪，
本文編號：2147641
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