小鼠抗人CD22單克隆抗體HIB22的生物學(xué)功能研究及其單鏈抗體的構(gòu)建
發(fā)布時(shí)間：2019-03-14 20:30
【摘要】： CD22是B細(xì)胞受體(BCR)的抑制共受體,在細(xì)胞調(diào)節(jié)抗原受體信號(hào)應(yīng)答的過程中必不可少。CD22只選擇性表達(dá)于正常和惡性的成熟B淋巴細(xì)胞系,可以在85%以上的B細(xì)胞非霍奇金淋巴瘤中檢測(cè)到;表達(dá)于成熟和惡性B細(xì)胞膜上的CD22與天然配體和抗體結(jié)合后迅速內(nèi)化;而且CD22在所有惡性細(xì)胞上的表達(dá)穩(wěn)定,不隨胞外環(huán)境不同而發(fā)生改變。這些優(yōu)勢(shì)使CD22成為治療NHL及自身免疫疾病的良好靶點(diǎn)。 針對(duì)CD22的靶向治療研究主要集中在對(duì)抗CD22單克隆抗體及其衍生藥物的開發(fā)上,其中Epratuzumab就是目前廣泛用于臨床試驗(yàn)的抗CD22單克隆抗體之一。HIB22為中國醫(yī)學(xué)科學(xué)院血液學(xué)研究所免疫室生產(chǎn)的鼠抗人CD22單克隆抗體,與Epratuzumab單抗識(shí)別位點(diǎn)不同。由于CD22可以通過配體依賴性與非依賴性機(jī)制調(diào)節(jié)B細(xì)胞作用,因此使用不同的單克隆抗體可以產(chǎn)生一系列臨床療效。 本研究運(yùn)用流式細(xì)胞術(shù)、MTT檢測(cè)、RT-PCR、共聚焦顯微鏡等方法對(duì)HIB22單抗做了如下體外生物學(xué)功能研究:HIB22單抗對(duì)NHL細(xì)胞系Raji、Daudi增殖及凋亡的影響;HIB單抗對(duì)PWM刺激后的正常人PBMC集落生長(zhǎng)及CD25表達(dá)的影響;HIB22與CD22抗原結(jié)合后內(nèi)化的觀察。本研究還利用小鼠雜交瘤細(xì)胞制備抗CD22單鏈抗體,流程如下:RT-PCR方法從HIB22雜交瘤中分別提取抗體輕、重鏈可變區(qū)基因;Overlap-PCR構(gòu)建HIB22-scFv融合基因并定向克隆入原核表達(dá)載體pET22b(+),構(gòu)建重組質(zhì)粒pET22b-HIB22-scFv;將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)~9后IPTG誘導(dǎo)表達(dá)HIB22-scFv;可溶性表達(dá)產(chǎn)物經(jīng)鎳柱純化后,通過流式細(xì)胞術(shù)檢測(cè)與CD22~+的Raji細(xì)胞系細(xì)胞膜CD22分子的結(jié)合活性及特異性。 HIB22單抗的體外生物學(xué)研究表明:HIB22能夠有效抑制NHL細(xì)胞系Daudi、Raji的增殖并抑制原癌基因c-myc的過度轉(zhuǎn)錄;HIB22能夠抑制美洲商陸絲裂原(PWM)刺激的正常人周血單個(gè)核細(xì)胞的集落生長(zhǎng),抑制PWM對(duì)正常人PBMC中B細(xì)胞的激活,且對(duì)CD4~+CD25~+及CD8~+CD25~+細(xì)胞也有一定影響;HIB22與CD22結(jié)合能夠內(nèi)化。在HIB22單抗的基因工程改造中,成功構(gòu)建融合蛋白原核表達(dá)載體pET22b-HIB22-scFv,轉(zhuǎn)化E.coli表達(dá)后獲得可溶性目的蛋白;生物學(xué)活性檢測(cè)表明所表達(dá)的HIB22-scFv單鏈抗體可以特異性結(jié)合CD22~+Raji細(xì)胞系,并且通過競(jìng)爭(zhēng)抑制實(shí)驗(yàn)證明HIB22-scFv單鏈抗體的結(jié)合表位與其親本單克隆抗體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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 才琳;抗黃曲霉毒素B1單克隆抗體和單鏈抗體的制備及活性研究[D];黑龍江八一農(nóng)墾大學(xué);2010年
，本文編號(hào)：2440344
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