人DR5胞外段基因的克
发布时间：2018-07-27 11:14
【摘要】： 背景 死亡受体5(death receptor , DR5)是肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand, TRAIL)的主要受体之一,DR5与TRAIL结合后可以通过其胞内的死亡结构域传递凋亡信号,从而引起细胞凋亡的发生,是激活凋亡途径的很好的靶标。TRAIL可引起许多肿瘤细胞系的凋亡,对绝大多数正常细胞不具有凋亡效应,被认为是癌症治疗的潜在的生物制剂。随着研究的深入,出现了关于TRAIL可以引起正常肝脏细胞的凋亡的报道。于是人们转入了对抗DR5单克隆抗体的研究上来。由于TRAIL的功能发挥主要靠DR5介导凋亡信号,如能得到没有肝脏细胞毒性又能活化DR5的单克隆抗体,将可以做为TRAIL的替代物进行药物研制。 目的 获得具有生物活性的人DR5胞外段蛋白,制备具有活性的抗DR5单克隆抗体。 方法 通过RT-PCR从Jurkat细胞中钓取DR5的胞外段基因(55-183位氨基酸),克隆到pGEM?-T Easy载体中,经核酸序列测定正确后,将其再次克隆入原核表达载体pET-30a中,在E.coli BL21 (DE3)实现蛋白表达。通过SDS-PAGE和Western blot鉴定表达产物。ELISA法分析其与抗DR5单克隆抗体(mAb)结合能力,用DR5胞外段竞争阻断TRAIL对Jurkat细胞生长的抑制。通过杂交瘤技术筛选抗DR5单克隆抗体(A6)。MTT法检测了A6对Jurkat细胞的生长抑制作用。吉姆萨染色、PI/Annexinv双染检测了A6对Jurkat的凋亡作用。通过竞争ELISA方法分析了A6和TRAIL的作用位点的异同。 结果 克隆到人DR5基因的胞外段基因,测序结果和报道的一致;构建了人DR5胞外段基因的原核表达载体并实现在E.coli BL21 (DE3)中大量表达;SDS-PAGE表明所表达蛋白与预期蛋白分子量一致;Western blot及ELISA的结果表明该蛋白能与抗DR5抗体反应,竞争阻断试验表明DR5胞外段可阻断TRAIL对Jurkat细胞生长的抑制。 成功制备了一株活性较好的抗DR5单克隆抗体-A6。MTT法显示了A6对Jurkat细胞的生长抑制作用,在25μg/mL的浓度下,作用16小时,可以达到对Jurkat细胞90㳠的生长抑制率。吉姆萨染色从形态上显示了Jurkat细胞的凋亡。PI/AnnexinⅤ双染分析显示:Jurkat细胞经过250 ng/mL浓度的A6作用12小时后,Jurkat细胞的凋亡率可以达到52.46㳠。用40倍浓度的TRAIL和A6做竞争ELISA,结果显示了TRAIL和A6的作用位点是不同的。 结论 (1)成功地制备了具有生物活性的DR5胞外区。 (2)制备了一株抗DR5单克隆抗体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，
本文编号：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，
本文编号：2147641
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