抗人DR5单克隆抗体的制备及抗肿瘤作用

发布时间：2018-03-29 01:30

本文选题：DR5　切入点：凋亡　出处：《河南大学》2008年硕士论文

【摘要】： 背景 TRAIL(TNF-related apoptosis-inducing ligand)是属于能够诱导细胞凋亡的TNF超家族成员。作为二型跨膜蛋白,它能够引起包括肿瘤细胞及一些转化细胞的凋亡。而DR5是TRAIL的重要受体之一,在TRAIL诱导细胞凋亡中起着重要的作用。DR5或DR4特异性的抗体的作用将优于外源性TRAIL的应用,因为一些肿瘤细胞的表面不仅有DR5/DR4,而且还有诱骗受体的表达,使TRAIL的作用发挥受到限制,但是特异性的DR5抗体不会影响它的凋亡诱导作用。针对能够特异的通过和死亡受体结合而向下传递凋亡信号,引起靶细胞的凋亡的特点,DR5抗体克服了TRAIL的不良作用,成为理想的候选者。尽管国内外已经制备出一些DR5的抗体,其中有一种抗体已经进入临床一期的实验阶段,但是,至今仍没有上市的有效药物。目的对已获得的小鼠抗人DR5单克隆抗体的特异性、抗体亚型以及生物学功能进行鉴定,并对抗体通过DR5进一步诱导肿瘤细胞凋亡的机制进行探讨。方法将表达载体pET30a/DR5转化到BL21,经0.1mM IPTG诱导表达,镍柱纯化后,经SDS-PAGE、Western-blot和蛋白质谱分析鉴定抗原蛋白。以表达纯化的DR5抗原蛋白免疫Balb/c小鼠,经融合、筛选及反复克隆化为基础(抗体制备过程由天广实生物技术有限公司完成),对获得的抗人DR5单抗-WD1进行鉴定,Western Blot和FACS方法检测单抗WD1与重组的DR5及膜型DR5的结合。MTT法检测WD1对几种细胞的生长抑制作用。吉姆萨染色、Annexin V/PI双染检测WD1对Jurkat的凋亡作用。通过将pcDNA3.1/DR4重组质粒转染至293T细胞以FACS检测WD1与DR4的交叉反应性。通过RT-PCR的方法钓取Jurkat和Daudi细胞的DR5胞内段蛋白序列分析细胞的突变情况,做DISC免疫沉淀实验分析抗体与DR5激活的关系。结果原核表达载体在BL21中实现大量表达,经SDS-PAGE和蛋白质谱分析鉴定,在19kD处有一明显的诱导表达带,其分子量大小与预期相符。Western-blot分析表明,该表达蛋白带可被抗人DR5抗体特异性的识别。为单抗的鉴定奠定了基础。成功制备了一株活性较好的抗DR5单克隆抗体-WD1。亚型分析WD1的重链为IgG1型,轻链为κ型。WD1可识别19KD的DR5单体蛋白,而且也识别37KD的DR5二聚体蛋白。FACS分析结果提示WD1可与多种细胞的膜型DR5分子特异性结合,而与DR4+/293T细胞无交叉反应。MTT结果表明WD1对Jurkat、Molt-4细胞增殖的抑制作用呈剂量依赖性;细胞形态学(吉姆萨染色)和FACS ( Annexin V/PI)分析结果证实WD1对Jurkat细胞具有诱导凋亡效应。通过RT-PCR的方法钓取Jurkat和Daudi细胞的DR5胞内段蛋白序列,经比对测序结果,两种细胞均与文献报道的DR5胞内段序列完全一致,这样就证实了细胞系本身未发生突变,不影响DR5的传导功能。而DISC的免疫沉淀实验显示WD1之所以能诱导Jurkat细胞的凋亡,是因为WD1作用Jurkat细胞后,可以募集FADD和caspase-8,形成DISC复合物,继而引起细胞凋亡的发生。结论 (1)获得具有生物活性的DR5胞外段蛋白。 (2)制备一株有凋亡活性的抗DR5单克隆抗体WD1。 (3)DISC复合物的形成是单克隆抗体WD1激活细胞DR5发生凋亡的原因。
[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 .

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R730.5

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