小鼠抗人CD22单克隆抗体HIB22的生物学功能研究及其单链抗体的构建
发布时间：2019-03-14 20:30
【摘要】： CD22是B细胞受体(BCR)的抑制共受体,在细胞调节抗原受体信号应答的过程中必不可少。CD22只选择性表达于正常和恶性的成熟B淋巴细胞系,可以在85%以上的B细胞非霍奇金淋巴瘤中检测到;表达于成熟和恶性B细胞膜上的CD22与天然配体和抗体结合后迅速内化;而且CD22在所有恶性细胞上的表达稳定,不随胞外环境不同而发生改变。这些优势使CD22成为治疗NHL及自身免疫疾病的良好靶点。 针对CD22的靶向治疗研究主要集中在对抗CD22单克隆抗体及其衍生药物的开发上,其中Epratuzumab就是目前广泛用于临床试验的抗CD22单克隆抗体之一。HIB22为中国医学科学院血液学研究所免疫室生产的鼠抗人CD22单克隆抗体,与Epratuzumab单抗识别位点不同。由于CD22可以通过配体依赖性与非依赖性机制调节B细胞作用,因此使用不同的单克隆抗体可以产生一系列临床疗效。 本研究运用流式细胞术、MTT检测、RT-PCR、共聚焦显微镜等方法对HIB22单抗做了如下体外生物学功能研究:HIB22单抗对NHL细胞系Raji、Daudi增殖及凋亡的影响;HIB单抗对PWM刺激后的正常人PBMC集落生长及CD25表达的影响;HIB22与CD22抗原结合后内化的观察。本研究还利用小鼠杂交瘤细胞制备抗CD22单链抗体,流程如下:RT-PCR方法从HIB22杂交瘤中分别提取抗体轻、重链可变区基因;Overlap-PCR构建HIB22-scFv融合基因并定向克隆入原核表达载体pET22b(+),构建重组质粒pET22b-HIB22-scFv;将重组质粒转化大肠杆菌BL21(DE3)~9后IPTG诱导表达HIB22-scFv;可溶性表达产物经镍柱纯化后,通过流式细胞术检测与CD22~+的Raji细胞系细胞膜CD22分子的结合活性及特异性。 HIB22单抗的体外生物学研究表明:HIB22能够有效抑制NHL细胞系Daudi、Raji的增殖并抑制原癌基因c-myc的过度转录;HIB22能够抑制美洲商陆丝裂原(PWM)刺激的正常人周血单个核细胞的集落生长,抑制PWM对正常人PBMC中B细胞的激活,且对CD4~+CD25~+及CD8~+CD25~+细胞也有一定影响;HIB22与CD22结合能够内化。在HIB22单抗的基因工程改造中,成功构建融合蛋白原核表达载体pET22b-HIB22-scFv,转化E.coli表达后获得可溶性目的蛋白;生物学活性检测表明所表达的HIB22-scFv单链抗体可以特异性结合CD22~+Raji细胞系,并且通过竞争抑制实验证明HIB22-scFv单链抗体的结合表位与其亲本单克隆抗体HIB22一致。
[Abstract]:CD22 is the inhibitory coreceptor of B cell receptor (BCR), which is essential in the regulation of antigen receptor signal response. CD22 is selectively expressed in normal and malignant mature B lymphocyte lines. It can be detected in more than 85% of B-cell non-Hodgkin's lymphoma. The CD22 expressed on mature and malignant B cell membranes was internalized rapidly after binding with natural ligands and antibodies, and the expression of CD22 in all malignant cells was stable and did not change with the different extracellular environment. These advantages make CD22 a good target for the treatment of NHL and autoimmune diseases. The targeted therapy for CD22 mainly focuses on the development of anti-CD22 monoclonal antibody and its derivatives. Epratuzumab is one of the anti-CD22 monoclonal antibodies widely used in clinical trials. HIB22 is a mouse anti-human CD22 monoclonal antibody produced in the Institute of Hematology, Chinese Academy of Medical Sciences, which is different from the Epratuzumab monoclonal antibody recognition site. Because CD22 can regulate the action of B cells through ligand-dependent and independent mechanisms, the use of different monoclonal antibodies can produce a series of clinical effects. In this study, flow cytometry, MTT and RT-PCR, confocal microscopy were used to study the biological function of HIB22 monoclonal antibody in vitro. The effects of HIB22 monoclonal antibody on proliferation and apoptosis of NHL cell line Raji,Daudi were studied. The effect of HIB monoclonal antibody on PBMC colony growth and CD25 expression in normal subjects stimulated by PWM, and the observation of HIB22 internalization after binding to CD22 antigen. In this study, mouse hybridoma cells were used to prepare anti-CD22 scFv. The procedure was as follows: the light and heavy chain variable region genes of antibody were extracted from HIB22 hybridoma by RT-PCR method; Overlap-PCR constructed HIB22-scFv fusion gene and cloned it into prokaryotic expression vector pET22b (), to construct recombinant plasmid pET22b-HIB22-scFv;. The recombinant plasmid was transformed into E. coli BL21 (DE3) ~ 9 and IPTG induced expression of HIB22-scFv; in E. coli BL21 (DE3) ~ 9. The soluble expression product was purified by nickel column, and the binding activity and specificity of the soluble expression product to the membrane CD22 molecule of CD22~ Raji cell line were detected by flow cytometry. The biological studies of HIB22 monoclonal antibody in vitro showed that HIB22 could effectively inhibit the proliferation of NHL cell line Daudi,Raji and inhibit the over-transcription of proto-oncogene c-myc. HIB22 could inhibit the colony growth of normal human blood mononuclear cells stimulated by pokeweed mitogen (PWM), inhibit the activation of B cells in normal human PBMC by PWM, and have some effects on CD4~ CD25~ and CD8~ CD25~ cells, and the binding of HIB22 to CD22 could internalize. In the genetic engineering of HIB22 monoclonal antibody, the fusion protein prokaryotic expression vector pET22b-HIB22-scFv, was successfully constructed and transformed into E.coli, and the soluble target protein was obtained. The biological activity test showed that the expressed HIB22-scFv could specifically bind to CD22~ Raji cell line, and the binding epitope of HIB22-scFv was consistent with its parental monoclonal antibody HIB22 by competitive inhibition assay.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2440344
[Abstract]:CD22 is the inhibitory coreceptor of B cell receptor (BCR), which is essential in the regulation of antigen receptor signal response. CD22 is selectively expressed in normal and malignant mature B lymphocyte lines. It can be detected in more than 85% of B-cell non-Hodgkin's lymphoma. The CD22 expressed on mature and malignant B cell membranes was internalized rapidly after binding with natural ligands and antibodies, and the expression of CD22 in all malignant cells was stable and did not change with the different extracellular environment. These advantages make CD22 a good target for the treatment of NHL and autoimmune diseases. The targeted therapy for CD22 mainly focuses on the development of anti-CD22 monoclonal antibody and its derivatives. Epratuzumab is one of the anti-CD22 monoclonal antibodies widely used in clinical trials. HIB22 is a mouse anti-human CD22 monoclonal antibody produced in the Institute of Hematology, Chinese Academy of Medical Sciences, which is different from the Epratuzumab monoclonal antibody recognition site. Because CD22 can regulate the action of B cells through ligand-dependent and independent mechanisms, the use of different monoclonal antibodies can produce a series of clinical effects. In this study, flow cytometry, MTT and RT-PCR, confocal microscopy were used to study the biological function of HIB22 monoclonal antibody in vitro. The effects of HIB22 monoclonal antibody on proliferation and apoptosis of NHL cell line Raji,Daudi were studied. The effect of HIB monoclonal antibody on PBMC colony growth and CD25 expression in normal subjects stimulated by PWM, and the observation of HIB22 internalization after binding to CD22 antigen. In this study, mouse hybridoma cells were used to prepare anti-CD22 scFv. The procedure was as follows: the light and heavy chain variable region genes of antibody were extracted from HIB22 hybridoma by RT-PCR method; Overlap-PCR constructed HIB22-scFv fusion gene and cloned it into prokaryotic expression vector pET22b (), to construct recombinant plasmid pET22b-HIB22-scFv;. The recombinant plasmid was transformed into E. coli BL21 (DE3) ~ 9 and IPTG induced expression of HIB22-scFv; in E. coli BL21 (DE3) ~ 9. The soluble expression product was purified by nickel column, and the binding activity and specificity of the soluble expression product to the membrane CD22 molecule of CD22~ Raji cell line were detected by flow cytometry. The biological studies of HIB22 monoclonal antibody in vitro showed that HIB22 could effectively inhibit the proliferation of NHL cell line Daudi,Raji and inhibit the over-transcription of proto-oncogene c-myc. HIB22 could inhibit the colony growth of normal human blood mononuclear cells stimulated by pokeweed mitogen (PWM), inhibit the activation of B cells in normal human PBMC by PWM, and have some effects on CD4~ CD25~ and CD8~ CD25~ cells, and the binding of HIB22 to CD22 could internalize. In the genetic engineering of HIB22 monoclonal antibody, the fusion protein prokaryotic expression vector pET22b-HIB22-scFv, was successfully constructed and transformed into E.coli, and the soluble target protein was obtained. The biological activity test showed that the expressed HIB22-scFv could specifically bind to CD22~ Raji cell line, and the binding epitope of HIB22-scFv was consistent with its parental monoclonal antibody HIB22 by competitive inhibition assay.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 才琳;抗黄曲霉毒素B1单克隆抗体和单链抗体的制备及活性研究[D];黑龙江八一农垦大学;2010年
，本文编号：2440344
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