非B细胞来源IgκV区保守序列特异性抗体的制备及鉴定
发布时间：2018-08-23 10:23
【摘要】： 免疫球蛋白(Ig)是重要的免疫分子,其两种存在方式分别为:膜型和分泌型,前者作为B淋巴细胞表面的抗原受体在抗原识别、细胞的活化、分化甚至程序性细胞死亡中起重要作用;后者主要存在与血液及其它体液中的,能够与相应抗原相结合发挥抗体的效应功能。经典的免疫学理论认为,Ig的来源为B淋巴细胞及浆细胞,Ig基因仅在B淋巴细胞发育过程中成功进行选择性重排,而在其它体细胞中该基因处于胚系状态,或者仅能发生不完全的重排,即不发生功能性基因重排,故不会有Ig分子的产生。 2003年,北京大学邱晓彦教授课题组在Cancer Research发表论文证实上皮来源肿瘤细胞可产生Ig分子,该工作被评为2004年中国医药研究领域十大新闻之一。其工作证明了除B淋巴细胞及浆细胞外,在一些非免疫细胞,如上皮来源的肿瘤细胞(如肺癌、卵巢癌、肠癌、鼻咽癌、乳腺癌、胃癌、胰腺癌等)及部分正常的上皮细胞内Ig基因同样存在功能性基因重排,并可以产生Ig分子,因此现将其称为非B细胞来源Ig(non B-Ig);此外,更重要的是该课题组通过对non B-Ig分子功能的研究,表现在基因及蛋白质水平抑制癌细胞Ig分子的表达或封闭其活性,则癌细胞增殖能力明显减弱。上述现象将有可能使人们重新定义Ig分子的来源及其潜在的、有别于经典Ig的生物学活性,具有重要的学术价值。同时,也将对肿瘤发生与发展的生物学研究以及以肿瘤细胞表达的Ig分子做为靶点的肿瘤生物治疗研究产生重要的影响。可喜的是,非B细胞能够产生Ig的现象已逐渐被国内外学者所证实与认同。 已知,B细胞来源的Ig具独特的结构和功能,主要表现在:其可变区(V区)识别特定的抗原决定簇,不同的B淋巴细胞克隆产生不同的IgV区结构,从而构成了Ig分子的多样性,使得免疫系统能够应对自然界不同的抗原;其恒定区与一组特定的分子(如Fc受体、信号传导分子、补体的级联成分)相结合,行使Ig分子的效应功能。根据恒定区的不同,B淋巴细胞产生的Ig分子根据重链不同被分为五类,分别是IgG、IgM、IgA、IgD和IgE;其轻链则分为κ与λ型。 B细胞来源的Ig分子的可变区序列呈高度多样性,这是构成抗体及BCR多样性的结构基础。对非B细胞来源的Ig基因研究结果显示,与经典的Ig分子相似,这些non B-Ig分子的重链及轻链编码基因均发生了典型的基因重排及转录。但在其V-D-J或V-J序列、体细胞突变特点和类别转换等方面均呈现出与经典Ig分子不同的特征。邱晓彦教授课题组借助激光显微切割(LCM)及RT-PCR技术,证明所有肿瘤细胞来源的Ig分子V-D-J或V-J片段组合方式有明显的倾向性。表现在同一个体的不同细胞克隆、甚至不同个体肿瘤来源的Ig序列之间高度同源,甚至完全相同,并且这些序列在B淋巴细胞表达的Ig数据库中均无记录,提示这些序列的确是肿瘤细胞产生的。更令人惊奇的是,所有肿瘤细胞均表达序列完全相同或极其相似的(个别碱基的替换)重排模式,即Vκ4—1/Jκ3型Igκ转录本(专利申请号为200510107833.9)。这一现象提示,这些序列特点与组织类型无关,它们可能在不同的组织中发挥着相似的作用。 根据上述发现,作为与北京大学邱晓彦教授的合作项目,本研究的主要目的是制备这种保守的κ链可变区抗体。我们根据其特有Vκ4—1/Jκ3氨基酸序列,对比胚系基因可变区序列,综合考虑氨基酸组成、疏水性、抗原性、表面暴露性等因素,利用生物信息学方法分析预测了Vκ4—1/Jκ3型Igκ两个较为特异的B细胞识别表位,设计并合成三个短肽AL13(序列ASINCKSSQRVSL)、QF20(QAEDVAVYYCQQYYDTPVTF),AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,即AL13的延长序列)。同时获得由邱晓彦教授课题组提供的原核表达Vκ4—1/Jκ3型Igκ纯化融合蛋白(GST-V_κ4-1-J_κ3)。本实验分别以合成肽AL13、QF20交联载体免疫家兔获得单表位特异性多克隆抗体;分别以合成肽AL13B-KLH、原核蛋白GST-V_κ4-1-J_κ3免疫小鼠获得系列单克隆抗体抗。再通ELISA、WesternBlot、流式细胞术等方法鉴定了抗体的效价和特异性,为后续研究提供了有效工具。 第一章Vκ4-1-Jκ3型IgκV区特异性单表位多抗的制备及鉴定 将合成肽AL13、QF20分别与载体蛋白交联制备免疫原,免疫新西兰大白兔获得相应抗血清,经ELISA检测其抗体滴度分别为0.3×10~(-5)和0.6×10~(-5)。运用亲和层析方法对兔血清进行纯化,得到anti-AL13和anti-QF20单表位特异性多抗。ELISA结果显示,所得抗体与合成肽特异性结合,但不与正常人IgG反应。为了进一步证明所得抗体的特异性,以原核表达蛋白GST-Vκ4-1-Jκ3(39kD)和V5-51-Vκ4-1-Jκ3-pRA(34kD)作为抗原进行Western Blot检测,结果显示anti-AL13和anti-QF20都能够正确识别含Vκ4-1-Jκ3序列的原核表达蛋白,在预期位置上都有明确的条带出现,但基本不与商品化的人IgG的轻链结合;用anti-AL13检测HT-29细胞中内源性Igκ的表达,结果显示anti-AL13的阳性信号主要在HT-29细胞不可溶成分中(仅少量的存在于可溶性组分中),这种与经典Ig分子完全不同的细胞内定位,提示其可能具有不同于经典Ig分子的生物学活性。 第二章Vκ4-1-Jκ3型IgκV区特异性单克隆抗体的制备及鉴定 以合成肽AL13B-KLH作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞融合制备杂交瘤,经合成肽和原核表达蛋白(GST-Vκ4-1-Jκ3)筛选和三次克隆化,并从制备的腹水中纯化单克隆抗体,筛选后共获得3株(6G5、3H9.2、5D3)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞;同时以原核重组蛋白(GST-Vκ4-1-Jκ3)作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞进行融合,经筛选、克隆化后获得1株(4E9)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞。这4株单抗的亚类分别为IgG1、IgG1、IgG1和IgG2b。将腹水单抗经辛酸—硫酸铵沉淀法纯化,并用改良过碘酸钠法对4株抗体标记了辣根过氧化物酶。ELISA鉴定结果显示所得单抗均与合成肽及原核表达蛋白(GST-Vκ4-1-Jκ3)发生特异性结合。对合成肽AL13B-KLH来源的3株单抗的近一步ELISA鉴定结果显示:这3株单抗与常用的载体蛋白KLH、BC、BSA、OVA均无交叉反应,同时与商品化的人IgG、羊IgG、兔IgG、鼠IgG均不反应;用流式细胞术鉴定上述单抗可与以人结肠癌细胞系HT-29和人肺癌细胞系A549的细胞内蛋白发生明显的特异性结合,验证了Vκ4-1-Jκ3型Igκ的胞内表达;与人末梢血淋巴细胞、单核细胞、粒细胞均有高比例的结合。鉴于人与小鼠Igκ的同源性高达70%,我们对靶抗原序列与小鼠IgκV区进行了比对,用流式细胞术证明上述4株单抗可与小鼠肿瘤细胞系的胞内蛋白有明显结合;与小鼠脾细胞的部分细胞膜结合。用上述4株单抗鉴定人和小鼠细胞的Vκ4-1-Jκ3型Igκ表达谱工作仍在进行中。 第三章抗蓝载体单克隆抗体的制备及特性鉴定 该部分是作为硕士研究生进入实验室初期进行免疫学技能训练时完成的一项工作,并得以发表。蓝载体(Blue Carrier,BC)是从软体动物Concholepasconcholepas中分离出的血蓝蛋白,是钥孔戚血蓝素(Keyhole limpethemocyanin,KLH)的一种类似物。BC的溶解性与均一性明显优于KLH,作为合成肽以及半抗原免疫时的载体蛋白已被成功用于多克隆抗体以及单克隆抗体的制备。但此前国内并无针对BC单抗的工作报道。本研究在制备抗短肽(以蓝载体为交联载体)单克隆抗体的同时,有目的地筛选了抗蓝载体单克隆抗体,所获单抗585只与蓝载体特异性结合,与钥孔戚血蓝素(KLH)、牛血清白蛋白(BSA)、鸡卵清白蛋白(OVA)等载体蛋白不发生交叉反应。该单抗可成为深入研究蓝载体抗原、以及短肽或半抗原交联抗原的相关研究的有效工具。
[Abstract]:Immunoglobulin (Ig) is an important immune molecule. It exists in two ways: membrane type and secretory type. The former plays an important role in antigen recognition, cell activation, differentiation and even programmed cell death as an antigen receptor on the surface of B lymphocyte; the latter mainly exists in blood and other body fluids and can be associated with the corresponding antigen phase. The classical immunological theory holds that Ig is derived from B lymphocytes and plasma cells, and the Ig gene is selectively rearranged only during the development of B lymphocytes, whereas in other somatic cells the gene is in embryonic state, or can only undergo incomplete rearrangement, i.e. no functional gene rearrangement. Therefore, there will be no generation of Ig molecules.
In 2003, Professor Qiu Xiaoyan of Peking University published a paper in Cancer Research confirming that epithelial-derived tumor cells can produce Ig molecules. The work was named one of the top ten news reports in the field of Chinese medicine research in 2004. The work demonstrated that in addition to B-lymphocytes and plasma cells, some non-immune cells, such as epithelial-derived tumor cells, such as Lung cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, breast cancer, gastric cancer, pancreatic cancer, etc.) and some normal epithelial cells also have functional gene rearrangement and can produce Ig molecules, so it is now called non-B-cell-derived Ig (non-B-Ig); moreover, more importantly, through the study of the function of non-B-Ig molecule, the team showed in the base. These phenomena will make it possible to redefine the origin and potential of Ig molecules, which are different from the biological activity of classical Ig and have important academic value. Researches and tumor biotherapy targeting Ig molecules expressed by tumor cells have important implications. Fortunately, the phenomenon that non-B cells can produce Ig has been gradually confirmed and recognized by scholars at home and abroad.
It is known that B-cell-derived Ig has a unique structure and function, mainly manifested in: its variable region (V region) recognizes specific antigen determinants, different B-lymphocyte clones produce different IgV region structure, thus constituting the diversity of Ig molecules, so that the immune system can respond to different antigens in nature; its constant region and a group of specific components. According to the constant region, the Ig molecules produced by B lymphocytes are classified into five categories according to the different heavy chains: IgG, IgM, IgA, IgD and IgE, and the light chains are classified into kappa and lambda.
Variable region sequences of B-cell-derived Ig molecules are highly diverse, which is the structural basis of antibody and BCR diversity. Similar to classical Ig molecules, the results of non-B-cell-derived Ig gene studies show that the heavy chain and light chain coding genes of these non-B-Ig molecules have undergone typical gene rearrangement and transcription, but in their V-D-J or V-J genes, both of them have undergone typical gene rearrangement and transcription. Professor Qiu Xiaoyan's research team demonstrated that the combination of V-D-J or V-J fragments of Ig molecules derived from all tumor cells had obvious tendency by means of laser microdissection (LCM) and RT-PCR. Highly homologous, or even identical, Ig sequences from different individual tumor sources were not recorded in the Ig database of B lymphocyte expression, suggesting that these sequences were indeed produced by tumor cells. More surprisingly, all tumor cells expressed identical or very similar sequences (individual bases). The pattern of rearrangement, i.e. V-kappa 4-1/J-kappa 3 Ig-kappa transcript (patent number 2005 10107833.9), suggests that these sequence characteristics are not related to the type of tissue, and they may play a similar role in different tissues.
According to the above findings, as a cooperative project with Professor Qiu Xiaoyan of Peking University, the main purpose of this study is to prepare the conserved variable region antibody of the kappa chain. Two specific B-cell recognition epitopes of Vkappa 4-1/J kappa 3 Ig kappa were analyzed and predicted by bioinformatics. Three short peptides, AL13 (ASINCKSSQRVSL), QF20 (QAEDVAVYCQYDTPVTF), AL13B (TQSPDSLVSLGERASINCKSSQRVSLG, or AL13 extension sequence), were designed and synthesized. The prokaryotic table provided by Professor Qiu Xiaoyan's research group was also obtained. The purified fusion protein (GST-V_kappa_4-1-J_kappa_3) was obtained by immunizing rabbits with synthetic peptides AL13 and QF20 respectively, and mice immunized with synthetic peptides AL13B-KLH and prokaryotic protein GST-V_kappa_4-1-J_kappa_3 were immunized with a series of monoclonal antibodies. The titers and specificities of the antibodies were identified by the method and so on, which provided an effective tool for further research.
Chapter one preparation and identification of V kappa 4-1-J kappa 3 Ig kappa V region specific single epitope polyclonal antibody
The synthetic peptides AL13 and QF20 were cross-linked with the carrier protein to prepare the immunogen. The corresponding antisera were obtained from New Zealand white rabbits immunized with these peptides. The titers of antibodies were 0.3 (-5) and 0.6 (-5) respectively by ELISA. The rabbit sera were purified by affinity chromatography, and anti-AL13 and anti-QF20 single epitope specific polyantibodies were obtained. To further prove the specificity of the antibody, the prokaryotic expression proteins GST-Vkappa 4-1-J kappa 3 (39kD) and V5-51-Vkappa 4-1-J kappa 3-pRA (34kD) were used as antigens for Western Blot assay. The results showed that both anti-AL13 and anti-QF20 could correctly recognize the Vkappa 4-1-J kappa 3 sequence. Prokaryotic expression proteins showed clear bands at expected sites, but did not bind to commercialized light chains of human IgG. Anti-AL13 was used to detect the expression of endogenous Ig-kappa in HT-29 cells. The results showed that the positive signal of anti-AL13 was mainly found in the insoluble components of HT-29 cells (only a small amount in the soluble components). Different intracellular localization of classical Ig molecules suggests that they may have different biological activities from classical Ig molecules.
The second chapter is the preparation and identification of V kappa 4-1-J type 3 Ig kappa V specific monoclonal antibody.
Balb/c mice were immunized with synthetic peptide AL13B-KLH as immunogen. The spleen cells were fused with NS-1 cells to prepare hybridoma. The synthesized peptide and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3) were screened and cloned for three times. Monoclonal antibodies were purified from the ascites. Three strains (6G5,3H9.2,5D3) secreted stably Ig kappa against Vkappa 4-1-J kappa 3. Balb/c mice were immunized with prokaryotic recombinant protein (GST-Vkappa 4-1-J kappa 3) as immunogen, and their spleen cells were fused with NS-1 cells. After screening and cloning, a hybridoma cell line (4E9) secreting antibodies against Vkappa 4-1-J kappa 3 Ig kappa stably was obtained. 2b. Ascites monoclonal antibodies were purified by octanoic acid-ammonium sulfate precipitation method and labeled with horseradish peroxidase by modified sodium periodate method. The results of ELISA identification showed that the monoclonal antibodies were specifically bound to synthetic peptides and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3). The results showed that the three monoclonal antibodies did not cross-react with carrier proteins KLH, BC, BSA and OVA, and did not react with commercialized human IgG, sheep IgG, rabbit IgG and mouse IgG. In view of the 70% homology between human and mouse Ig-kappa, we compared the target antigen sequence with mouse Ig-kappa V region and confirmed that the four McAbs could bind to the intracellular protein of mouse tumor cell line by flow cytometry. The identification of Vkappa 4-1-J kappa 3 Ig kappa expression profiles in human and mouse cells by these four monoclonal antibodies is still in progress.
The third chapter is preparation and characterization of monoclonal antibodies against blue carriers.
This part is a job done as a postgraduate student in the early stage of the laboratory immunological skills training, and published. Blue Carrier (BC) is a hemocyanin isolated from the mollusk Concholepas concholepas, is a key hole limpethemocyanin (KLH) analogue. As a carrier protein for synthetic peptides and hapten immunization, it has been successfully used in the preparation of polyclonal antibodies and monoclonal antibodies. However, there have been no reports on the preparation of monoclonal antibodies against BC. In this study, monoclonal antibodies against short peptides (using blue carriers as cross-linking carriers) were prepared and screened purposefully. A total of 585 monoclonal antibodies against blue vectors were obtained. The monoclonal antibodies specifically bind to blue vectors and do not cross-react with carrier proteins such as key hole hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA). The monoclonal antibodies can be used as an effective tool for further study of blue vectors antigens, short peptides or hapten cross-linked antigens. It is.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2198781
[Abstract]:Immunoglobulin (Ig) is an important immune molecule. It exists in two ways: membrane type and secretory type. The former plays an important role in antigen recognition, cell activation, differentiation and even programmed cell death as an antigen receptor on the surface of B lymphocyte; the latter mainly exists in blood and other body fluids and can be associated with the corresponding antigen phase. The classical immunological theory holds that Ig is derived from B lymphocytes and plasma cells, and the Ig gene is selectively rearranged only during the development of B lymphocytes, whereas in other somatic cells the gene is in embryonic state, or can only undergo incomplete rearrangement, i.e. no functional gene rearrangement. Therefore, there will be no generation of Ig molecules.
In 2003, Professor Qiu Xiaoyan of Peking University published a paper in Cancer Research confirming that epithelial-derived tumor cells can produce Ig molecules. The work was named one of the top ten news reports in the field of Chinese medicine research in 2004. The work demonstrated that in addition to B-lymphocytes and plasma cells, some non-immune cells, such as epithelial-derived tumor cells, such as Lung cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, breast cancer, gastric cancer, pancreatic cancer, etc.) and some normal epithelial cells also have functional gene rearrangement and can produce Ig molecules, so it is now called non-B-cell-derived Ig (non-B-Ig); moreover, more importantly, through the study of the function of non-B-Ig molecule, the team showed in the base. These phenomena will make it possible to redefine the origin and potential of Ig molecules, which are different from the biological activity of classical Ig and have important academic value. Researches and tumor biotherapy targeting Ig molecules expressed by tumor cells have important implications. Fortunately, the phenomenon that non-B cells can produce Ig has been gradually confirmed and recognized by scholars at home and abroad.
It is known that B-cell-derived Ig has a unique structure and function, mainly manifested in: its variable region (V region) recognizes specific antigen determinants, different B-lymphocyte clones produce different IgV region structure, thus constituting the diversity of Ig molecules, so that the immune system can respond to different antigens in nature; its constant region and a group of specific components. According to the constant region, the Ig molecules produced by B lymphocytes are classified into five categories according to the different heavy chains: IgG, IgM, IgA, IgD and IgE, and the light chains are classified into kappa and lambda.
Variable region sequences of B-cell-derived Ig molecules are highly diverse, which is the structural basis of antibody and BCR diversity. Similar to classical Ig molecules, the results of non-B-cell-derived Ig gene studies show that the heavy chain and light chain coding genes of these non-B-Ig molecules have undergone typical gene rearrangement and transcription, but in their V-D-J or V-J genes, both of them have undergone typical gene rearrangement and transcription. Professor Qiu Xiaoyan's research team demonstrated that the combination of V-D-J or V-J fragments of Ig molecules derived from all tumor cells had obvious tendency by means of laser microdissection (LCM) and RT-PCR. Highly homologous, or even identical, Ig sequences from different individual tumor sources were not recorded in the Ig database of B lymphocyte expression, suggesting that these sequences were indeed produced by tumor cells. More surprisingly, all tumor cells expressed identical or very similar sequences (individual bases). The pattern of rearrangement, i.e. V-kappa 4-1/J-kappa 3 Ig-kappa transcript (patent number 2005 10107833.9), suggests that these sequence characteristics are not related to the type of tissue, and they may play a similar role in different tissues.
According to the above findings, as a cooperative project with Professor Qiu Xiaoyan of Peking University, the main purpose of this study is to prepare the conserved variable region antibody of the kappa chain. Two specific B-cell recognition epitopes of Vkappa 4-1/J kappa 3 Ig kappa were analyzed and predicted by bioinformatics. Three short peptides, AL13 (ASINCKSSQRVSL), QF20 (QAEDVAVYCQYDTPVTF), AL13B (TQSPDSLVSLGERASINCKSSQRVSLG, or AL13 extension sequence), were designed and synthesized. The prokaryotic table provided by Professor Qiu Xiaoyan's research group was also obtained. The purified fusion protein (GST-V_kappa_4-1-J_kappa_3) was obtained by immunizing rabbits with synthetic peptides AL13 and QF20 respectively, and mice immunized with synthetic peptides AL13B-KLH and prokaryotic protein GST-V_kappa_4-1-J_kappa_3 were immunized with a series of monoclonal antibodies. The titers and specificities of the antibodies were identified by the method and so on, which provided an effective tool for further research.
Chapter one preparation and identification of V kappa 4-1-J kappa 3 Ig kappa V region specific single epitope polyclonal antibody
The synthetic peptides AL13 and QF20 were cross-linked with the carrier protein to prepare the immunogen. The corresponding antisera were obtained from New Zealand white rabbits immunized with these peptides. The titers of antibodies were 0.3 (-5) and 0.6 (-5) respectively by ELISA. The rabbit sera were purified by affinity chromatography, and anti-AL13 and anti-QF20 single epitope specific polyantibodies were obtained. To further prove the specificity of the antibody, the prokaryotic expression proteins GST-Vkappa 4-1-J kappa 3 (39kD) and V5-51-Vkappa 4-1-J kappa 3-pRA (34kD) were used as antigens for Western Blot assay. The results showed that both anti-AL13 and anti-QF20 could correctly recognize the Vkappa 4-1-J kappa 3 sequence. Prokaryotic expression proteins showed clear bands at expected sites, but did not bind to commercialized light chains of human IgG. Anti-AL13 was used to detect the expression of endogenous Ig-kappa in HT-29 cells. The results showed that the positive signal of anti-AL13 was mainly found in the insoluble components of HT-29 cells (only a small amount in the soluble components). Different intracellular localization of classical Ig molecules suggests that they may have different biological activities from classical Ig molecules.
The second chapter is the preparation and identification of V kappa 4-1-J type 3 Ig kappa V specific monoclonal antibody.
Balb/c mice were immunized with synthetic peptide AL13B-KLH as immunogen. The spleen cells were fused with NS-1 cells to prepare hybridoma. The synthesized peptide and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3) were screened and cloned for three times. Monoclonal antibodies were purified from the ascites. Three strains (6G5,3H9.2,5D3) secreted stably Ig kappa against Vkappa 4-1-J kappa 3. Balb/c mice were immunized with prokaryotic recombinant protein (GST-Vkappa 4-1-J kappa 3) as immunogen, and their spleen cells were fused with NS-1 cells. After screening and cloning, a hybridoma cell line (4E9) secreting antibodies against Vkappa 4-1-J kappa 3 Ig kappa stably was obtained. 2b. Ascites monoclonal antibodies were purified by octanoic acid-ammonium sulfate precipitation method and labeled with horseradish peroxidase by modified sodium periodate method. The results of ELISA identification showed that the monoclonal antibodies were specifically bound to synthetic peptides and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3). The results showed that the three monoclonal antibodies did not cross-react with carrier proteins KLH, BC, BSA and OVA, and did not react with commercialized human IgG, sheep IgG, rabbit IgG and mouse IgG. In view of the 70% homology between human and mouse Ig-kappa, we compared the target antigen sequence with mouse Ig-kappa V region and confirmed that the four McAbs could bind to the intracellular protein of mouse tumor cell line by flow cytometry. The identification of Vkappa 4-1-J kappa 3 Ig kappa expression profiles in human and mouse cells by these four monoclonal antibodies is still in progress.
The third chapter is preparation and characterization of monoclonal antibodies against blue carriers.
This part is a job done as a postgraduate student in the early stage of the laboratory immunological skills training, and published. Blue Carrier (BC) is a hemocyanin isolated from the mollusk Concholepas concholepas, is a key hole limpethemocyanin (KLH) analogue. As a carrier protein for synthetic peptides and hapten immunization, it has been successfully used in the preparation of polyclonal antibodies and monoclonal antibodies. However, there have been no reports on the preparation of monoclonal antibodies against BC. In this study, monoclonal antibodies against short peptides (using blue carriers as cross-linking carriers) were prepared and screened purposefully. A total of 585 monoclonal antibodies against blue vectors were obtained. The monoclonal antibodies specifically bind to blue vectors and do not cross-react with carrier proteins such as key hole hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA). The monoclonal antibodies can be used as an effective tool for further study of blue vectors antigens, short peptides or hapten cross-linked antigens. It is.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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