人ICOS分子的表达与单克隆抗体的研制及其生物学功能的研究

发布时间：2018-04-12 16:18

本文选题：ICOS + 分子　；参考：《苏州大学》2003年博士论文

【摘要】： ICOS(Inducible co-stimulator)是最近发现的CD28家族的新分子，主要表达在活化的T细胞上，与表达在B细胞、巨噬细胞或树突状细胞表面的相应配体GL50分子相互作用，在机体的再次免疫阶段、炎症反应、自身免疫性疾病、肿瘤免疫和移植免疫中发挥了重要作用，展示了其潜在的应用前景。然而，ICOS-GL50在机体免疫调节中的分子机制尚待进一步阐明，更重要的是，目前对ICOS-GL50的功能研究主要集中在小鼠。鉴此，本研究在国内率先采用基因工程技术克隆了全长的人ICOS基因和胞外段cDNA，构建了ICOS转基因细胞，并在大肠杆菌中成功表达了具有生物学活性的ICOS可溶性重组蛋白，对其生物学功能作了初步探讨。利用ICOS转基因细胞作免疫原，成功获得两株鼠抗人ICOS单克隆抗体，并对其中一株的生物学特性作了鉴定。在此基础上，分析了ICOS-GL50在DC成熟与体液免疫中的作用，并进一步研究了ICOS-GL50信号对多发性骨髓瘤细胞体内外生物学行为的作用及其可能机制。一、人ICOS分子的表达及其生物学活性研究 1、人ICOS的基因克隆及其在大肠杆菌中的表达采用RT-PCR法从活化的人T细胞中扩增了ICOS全长基因，装入T-vector克隆载体。重组质粒T-ICOS经测序确证后，进而PCR扩增ICOS胞外段编码cDNA，即可溶性的ICOS基因片段，并克隆入表达载体pET-28a。构建的重组表达载体pET-ICOS经酶切和序列分析证实后，转化BL-21大肠杆菌，在1～5mmol／L IPTG诱人Icos分子的表达与单克隆抗体的研制及其生物学功能的研究中文摘要导下，重组蛋白以包涵体方式在BL一21菌株中诱导表达。分离的包涵体经变性、复性和FPLC分离纯化后即获得了较高纯度的ICOS重组蛋白。 2、L929一ICOS转基因细胞的建立合成全长ICOS(包括引导肤序列)特异性引物，引物中分别引入刀。朋Hl与Ec口R I酶切位点，以T-ICOS质粒为模板进行PCR反应。PCR产物经刀口脚Hl与Ec口Rl 双酶切后装入逆转录病毒表达载体pGEZ舰A一介nn，，测序正确的重组表达载体 pGEZ一ICOS与辅助病毒载体pHIT6O和pHIT456混合后，脂质体法转染包装细胞293T 细胞，以获得具有感染能力的重组逆转录病毒，后者再感染L929细胞于6孔板中，在zcocin(500林留ml)加压筛选获得抗性转基因细胞的基础上，采用免疫荧光标记和流式细胞仪分选，即获得了高表达ICOS的转基因L929细胞。 3、ICOS蛋白抑制Daudi细胞的生长和增殖我们检测了部分人的恶性淋巴瘤细胞株的GL50表达，初步结果表明，Daudi、 HL6O细胞高表达该分子，其阳性表达率分别为99.1%、80.1%。为了观察Daudi细胞高表达GLSO分子的生物学意义，将L929一ICOS转基因细胞、可溶性ICOS分别与 Daudi细胞体外共培养后，分析了Daudi细胞的生长抑制及凋亡效应。当培养24小时，显微镜下两组均见Daudi细胞聚集成团，细胞中出现颗粒，折光性和立体感减弱。同时，Daudi细胞的表型也明显发生了变化，导致粘附分子CD54、趋化因子CXCR4 下调，Fas表达水平有不同程度的升高。通过细胞计数、流式细胞仪分析证实 L929一ICOS转基因细胞对肿瘤生长的具有显著的抑制作用，并能促使细胞凋亡，可溶性ICOS重组蛋白也有类似的作用，并且对Daudi促凋亡的效应高于转基因细胞。另外，研究还发现，大多数高表达GL50分子肿瘤细胞，同时也异常表达了B7、CD40 分子，它们之间有无内在联系?这种现象与恶性B细胞的生物学行为有何关联?值得我们进一步的研究。综合本研究结果:我们成功地在大肠杆菌中表达出ICOS重组蛋白，并构建了高表达膜型ICOS分子的转基因细胞，对其生物学功能研究表明，ICOS转基因细胞和重组蛋白均能直接并显著地抑制恶性淋巴瘤Daudi的体外增殖，畴导其凋亡，故重组ICOS蛋白在恶性B细胞肿瘤的生物治疗中可能具有潜在的运用价值。人IcOS分子的表达与单克隆抗体的研制及其生物学功能的研究中文摘要二、ICoS一GL5o在DC成熟及T细胞依赖性B细胞产生抗体中的作用 1、ICoS一GL50在诱导DC分化成熟中的作用树突状细胞(DC)是体内己知功能最强的专职性抗原递呈细胞(妙C)，能摄取、加工及递呈抗原，并能刺激未致敏T细胞的增殖和活化，从而启动机体的特异性免疫应答。对骨髓、脐血来源的CD34+造血干细胞的研究发现，CD34+造血干细胞不表达GL50，但是经过TNF一。、GM一CSF刺激后，可以在12h内诱导上调表达GL50，值得注意的是，GL50先于CD80/C D86被诱导表达，提示GL50可能是单核向DC 成熟发展过程的早期分化指标之一。目前仅了解DC表达GL50，但是对于其是否在 DC的分化成熟以及增殖方面起作用，至今还没有报道。为了探索GL50在DC上表达的意义，我们用ICOS转基因细胞和可溶性ICOS蛋白初步研究了其在DC的成熟中的作用，研究结果显示:在经GM一CSF+IL一4诱导的同时加入ICOS蛋白，可以诱导单核细胞向不成熟的DC分化过程中上调CD40、CD83，在低剂量CD40 mAb进一步的刺激下，可以发育成为成熟的DC，值得关注的是，成熟的DC细胞的CXCR4、 CDla、CDI lb下调，而CD45RA上调。我们推测ICOS一GL50早期激发后，有可能使不成熟的DC向Ix型的DC
[Abstract]:ICOS (Inducible co-stimulator) is a recently discovered new molecules of CD28 family, mainly expressed in activated T cells, and its expression in B cells, the interaction of macrophages or dendritic cells on the surface of the corresponding ligand GL50 molecules in the body of the second immune stage, inflammation, autoimmune diseases, has played an important role in tumor immunity and transplantation immunity, demonstrating its potential applications. However, the molecular mechanism of ICOS-GL50 in immune regulation remains to be further elucidated, more importantly, the functional study of ICOS-GL50 mainly concentrated in mice. In view of this, this study first in China using genetic engineering technology to clone full length people ICOS gene and extracellular cDNA, constructed the ICOS transgenic cells, and expressed in E.coli successfully ICOS with biological activity of soluble recombinant protein, the biological function of the early To use ICOS as immunogen. Transgenic cells, successfully obtained two strains of mouse anti human ICOS monoclonal antibody, and the biological characteristics of one strain were identified. Based on the analysis of the role of ICOS-GL50 in DC maturation and humoral immunity, and further study the role of ICOS-GL50 signaling in multiple myeloma in vitro and in vivo biological behavior and its possible mechanism.
Expression and biological activity of human ICOS molecule
1, gene cloning of human ICOS and its expression in Escherichia coli
Using RT-PCR amplified the full-length ICOS gene from human T cell activation, cloned into T-vector plasmid. The recombinant plasmid T-ICOS by sequencing, and PCR amplification of ICOS extracellular fragment encoding cDNA gene fragment of ICOS, can be dissolved, and cloned into the expression vector pET-ICOS recombinant expression confirmed by enzyme digestion and sequence analysis vector pET-28a. was constructed and transformed into Escherichia coli BL-21. In 1 ~ 5mmol / L induced IPTG
Study on the expression of human Icos molecules and the development of monoclonal antibodies and their biological functions
Chinese abstract
Under the guidance of the recombinant protein, the expression of the inclusion body was induced in the BL 21 strain. The isolated inclusion body was denatured and reproduced.
High purity recombinant protein of ICOS was obtained after separation and purification of sex and FPLC.
The establishment of 2, L929 ICOS transgenic cells
The specific primers of the full length ICOS (including the guided skin sequence) were synthesized. The primers were introduced into the primers. Hl and Ec mouth R were introduced.
I enzyme cutting site and T-ICOS plasmid as template for PCR reaction of.PCR products via Hl and Ec mouth Rl
Double enzyme was loaded into retroviral vector pGEZ, A a NN, and the correct recombinant expression vector was sequenced
PGEZ 1 ICOS is mixed with pHIT6O and pHIT456, an auxiliary viral vector, and transfected 293T by liposome
Cells to obtain recombinant retrovirus infection ability, which infected L929 cells in 6 well plates,
Immunofluorescence markers were used on the basis of zcocin (500 Lin stay ml) pressure screening for resistant transgenic cells.
With the flow cytometry, the transgenic L929 cells with high expression of ICOS were obtained.
3, ICOS protein inhibits the growth and proliferation of Daudi cells
We detected the expression of GL50 in some human lymphoma cell lines, and the preliminary results showed that Daudi,
The positive expression rate of HL6O cells was 99.1%, respectively, and 80.1%. was used to observe Daudi fine.
The biological significance of high expression of GLSO molecules, L929 1 ICOS transgenic cells, soluble ICOS and
After co culture of Daudi cells in vitro, the growth inhibition and apoptosis effect of Daudi cells were analyzed. When 24 small cells were cultured, the cells were cultured.
At the time, two groups of Daudi cells were clustered together under the microscope, and there were particles, refraction and stereotyping in the cells.
At the same time, the phenotype of Daudi cells also changed significantly, leading to the adhesion molecule CD54, chemokine CXCR4
Down regulation, the expression level of Fas is elevated in varying degrees. By cell count, flow cytometry analysis confirmed
L929 - ICOS transgenic cells have a significant inhibitory effect on tumor growth and can induce apoptosis.
The recombinant protein of soluble ICOS also has a similar effect, and the effect of Daudi on apoptosis is higher than that of transgenic cells.
In addition, the study also found that most of the high expression of GL50 molecular tumor cells, but also abnormal expression of B7, CD40
Are there any intrinsic connections between them? What is the correlation between this phenomenon and the biological behavior of malignant B cells?
We'll have to study further.
Combined with the results of this study, we successfully expressed ICOS recombinant protein in Escherichia coli and constructed a high level of recombinant protein.
Transgenic cells expressing the membrane type ICOS molecules, and their biological function studies show that the ICOS transgenic cells and the cells are genetically modified.
The recombinant protein can directly and significantly inhibit the proliferation of malignant lymphoma Daudi in vitro, and the domain leads to its apoptosis.
Group ICOS protein may have potential application value in the biological treatment of malignant B cell tumor.
The expression of human IcOS molecules and the development of monoclonal antibodies and their biological functions
Two, the role of ICoS 1 GL5o in the production of antibodies in DC mature and T cell dependent B cells
The role of 1, ICoS 1 GL50 in the induction of DC differentiation and maturation
Dendritic cells (DC) are the best known specific antigen presenting cells (C) that have the strongest known function in the body.
Processing and presenting antigen, and stimulating the proliferation and activation of the non sensitized T cells, thus triggering the specific immunity of the body.
A study of CD34+ hematopoietic stem cells derived from bone marrow and umbilical cord blood found that CD34+ hematopoietic stem cells do not
GL50 is expressed, but after TNF 1. After GM CSF stimulation, the expression of GL50 can be up-regulated in 12h.
It is worth noting that GL50 was induced earlier than CD80/C D86, suggesting that GL50 may be mononuclear to DC
One of the early differentiation indicators of the mature development process. At present, only DC is known to express GL50, but whether it is in it or not
DC plays a role in differentiation and proliferation and has not yet been reported. In order to explore GL50 on the DC table
To the significance of this, we preliminarily studied the maturation of DC by using ICOS transgenic cells and soluble ICOS protein.
The results of the study show that the addition of ICOS protein at the same time as GM CSF+IL 1 4 can be induced.
In the process of immature DC differentiation, mononuclear cells increase CD40, CD83, and enter low dose CD40 mAb.
A step of stimulation, can develop into a mature DC, and it is noteworthy that the CXCR4 of the mature DC cells,
CDla, CDI LB down, and CD45RA up-regulated. We speculate that after ICOS GL50 early excitation, it is possible
Make the immature DC to Ix type DC

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2003
【分类号】：R392.1

【引证文献】