技术领域technical field
本发明涉及一种单克隆抗体，具体涉及抗人DLL4单克隆抗体6F12的制备方法。The invention relates to a monoclonal antibody, in particular to a preparation method of the anti-human DLL4 monoclonal antibody 6F12.
背景技术Background technique
Notch信号通路是进化上高度保守的一套信号系统，在细胞增殖、分化和凋亡，以及细胞生长和各种生理功能中均发挥重要作用。Notch信号分子在绝大多数的多细胞生物体内表达。哺乳动物主要表达四种Notch受体（分别为：Notch1、2、3、4）和五种Notch配体（分别为：DLL1、DLL3、DLL4、Jagged1和Jagged2）。 The Notch signaling pathway is a set of highly conserved signaling systems in evolution, which play an important role in cell proliferation, differentiation and apoptosis, as well as cell growth and various physiological functions. Notch signaling molecules are expressed in most multicellular organisms. Mammals mainly express four Notch receptors (respectively: Notch1, 2, 3, 4) and five Notch ligands (respectively: DLL1, DLL3, DLL4, Jagged1 and Jagged2).
早期研究显示，DLL4在血管内皮细胞内高度选择性表达，对于调控内皮细胞发育至关重要。2004年Amsen及其同事发现通过LPS刺激包含有抗原递呈细胞的骨髓细胞可以诱导DLL4的表达。2007年，Skokos及其同事报道，CD8-DC通过DLL4分子激活Notch信号通路的方式可以诱导细胞向Th1方向分化，而这种方式是非IL-12依赖性的。随后有研究表明，在实验小鼠模型中，DLL4可以调控许多疾病的发病，例如炎症性疾病、呼吸道病毒感染、实验性过敏性结肠炎、实验性自身免疫性脑脊髓炎以及分支杆菌引起的肺肉芽肿。Zhang Yi教授的研究证实了表达DLL4的小鼠树突状细胞（dendritic cell, DCs）可以提高自身反应性T细胞的应答并介导移植物抗宿主反应。但是对人DLL4+ DCs的研究还有待进一步开展。Early studies have shown that DLL4 is highly selectively expressed in vascular endothelial cells and is critical for regulating endothelial cell development. In 2004, Amsen and colleagues found that stimulation of bone marrow cells containing antigen-presenting cells by LPS could induce the expression of DLL4. In 2007, Skokos and colleagues reported that CD8-DCs can induce cells to differentiate into Th1 by activating the Notch signaling pathway through DLL4 molecules, and this method is independent of IL-12. Subsequent studies have shown that in experimental mouse models, DLL4 can regulate the pathogenesis of many diseases, such as inflammatory diseases, respiratory viral infections, experimental allergic colitis, experimental autoimmune encephalomyelitis, and mycobacterial-induced lung disease. Granuloma. Professor Zhang Yi's research confirmed that mouse dendritic cells (DCs) expressing DLL4 can enhance the response of autoreactive T cells and mediate graft-versus-host response. But the research on human DLL4+ DCs needs further development.
最近，Zhang Yi教授等人的研究报道了人DLL4+ DCs在调节T细胞向Th1和Th17分化中的关键作用。来自于健康人的外周血中的CD1C+ DCs和浆细胞样DC（pDC）表面不表达DLL4分子。相比之下，进行同种异体造血干细胞移植的患者外周血中的DLL4+ CD1C+ DCs表达的DLL4 mRNA水平比健康人高16倍。在激活TLR信号后，来自于健康人的CD1C+ DCs表达的DLL4水平显著上调。相比之下pDCs上调的程度较低。活化后的DLL4+ DCs比未刺激的DCs能够更好地促进Th1和Th17分化。在DLL4+ DCs刺激活化T细胞的过程中，使用DLL4的中和抗体来阻断Notch信号，可以减少Th1和Th17细胞的产生。因此对于人外周血循环的DCs来说，DLL4是其诱导T细胞向Th1和Th17分化的一个重要功能性分子。这些发现为人类炎症性疾病的治疗和干预提供了可能的途径。Recently, the study by Prof. Zhang Yi et al. reported the key role of human DLL4+ DCs in regulating T cell differentiation towards Th1 and Th17. CD1C+ DCs and plasmacytoid DCs (pDCs) in peripheral blood from healthy individuals do not express DLL4 molecules on their surface. In contrast, DLL4+ CD1C+ DCs in the peripheral blood of patients undergoing allogeneic hematopoietic stem cell transplantation expressed 16-fold higher levels of DLL4 mRNA than healthy individuals. CD1C+ DCs from healthy individuals expressed significantly upregulated levels of DLL4 after activation of TLR signaling. In contrast, pDCs were less upregulated. Activated DLL4+ DCs can promote Th1 and Th17 differentiation better than unstimulated DCs. During the stimulation of activated T cells by DLL4+ DCs, blocking Notch signaling with DLL4 neutralizing antibodies can reduce the generation of Th1 and Th17 cells. Therefore, for DCs circulating in human peripheral blood, DLL4 is an important functional molecule for inducing T cells to differentiate into Th1 and Th17. These findings provide possible avenues for the treatment and intervention of human inflammatory diseases.
DLL4分子自被发现以来一直被广大科研工作者所关注，针对人DLL4分子也展开了许多方面的功能性研究。在许多功能性研究中，需要通过抗体标记后使用流式细胞仪将DLL4+的目的细胞分选出来。但目前使用的商品化抗人DLL4单克隆抗体还存在一定的局限性。目前许多知名抗体公司用于流式检测的抗人DLL4荧光抗体仅有一个克隆号（MHD4-46），而该克隆号的抗体在Zhang Yi教授发表的文献中明确报道了其具有阻断DLL4信号的功能。目前并没有一株商品化的单克隆抗体可用于标记并通过流式细胞仪分选出DLL4+ DCs的同时保留DLL4的信号功能。因此，研制抗人DLL4+功能性单克隆抗体将有助于更好的研究DLL4分子的生物学功能，为研究提供一种新的手段。Since the DLL4 molecule was discovered, it has been concerned by many scientific researchers, and many functional studies have been carried out on the human DLL4 molecule. In many functional studies, DLL4+ target cells need to be sorted out by flow cytometry after antibody labeling. However, the currently used commercial anti-human DLL4 monoclonal antibodies still have certain limitations. At present, many well-known antibody companies have only one clone number (MHD4-46) for the anti-human DLL4 fluorescent antibody used in flow cytometry, and the antibody with this clone number has been clearly reported in the literature published by Professor Zhang Yi that it can block DLL4 signal function. Currently, there is no commercially available monoclonal antibody that can be used to label and sort DLL4+ DCs by flow cytometry while retaining the signaling function of DLL4. Therefore, the development of anti-human DLL4+ functional monoclonal antibodies will help to better study the biological functions of DLL4 molecules and provide a new means for research.
发明内容Contents of the invention
本发明的发明目的是提供一种抗人DLL4单克隆抗体6F12的制备方法，由抗人DLL4单克隆抗体的杂交瘤细胞株产生。The object of the present invention is to provide a preparation method of anti-human DLL4 monoclonal antibody 6F12, which is produced by a hybridoma cell line of anti-human DLL4 monoclonal antibody.
为达到上述发明目的，本发明采用的技术方案是：In order to achieve the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:
抗人DLL4单克隆抗体6F12的制备方法，所述抗人DLL4单克隆抗体6F12由杂交瘤细胞株制备。The preparation method of the anti-human DLL4 monoclonal antibody 6F12, the anti-human DLL4 monoclonal antibody 6F12 is prepared by a hybridoma cell line.
上述技术方案中，所述杂交瘤细胞株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏号为CGMCC No.14284。In the above technical scheme, the hybridoma cell line is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 14284.
上述技术方案中，在杂交瘤培养液中接种杂交瘤细胞株，培养后将培养液分离纯化制备单克隆抗体。In the above technical solution, the hybridoma cell line is inoculated in the hybridoma culture fluid, and after culturing, the culture fluid is separated and purified to prepare monoclonal antibodies.
上述技术方案中，在动物腹腔内接种杂交瘤细胞株，将动物腹水液分离、纯化制备单克隆抗体。In the above technical scheme, the hybridoma cell line is inoculated in the peritoneal cavity of the animal, and the ascites fluid of the animal is separated and purified to prepare the monoclonal antibody.
本发明还公开了一种检测DLL4蛋白的表达水平的试剂或者分选DLL4+DC的试剂的制备方法，在杂交瘤培养液中接种杂交瘤细胞株，培养后将培养液分离纯化制备单克隆抗体；将所述单克隆抗体与分散介质混合制备得到检测DLL4蛋白的表达水平的试剂或者分选DLL4+DC的试剂。The invention also discloses a preparation method of a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+DC, inoculating hybridoma cell strains in the hybridoma culture medium, and separating and purifying the culture medium after culturing to prepare monoclonal antibodies ; Mix the monoclonal antibody with the dispersion medium to prepare a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+DC.
上述技术方案中，所述分散介质包括缓冲液。In the above technical solution, the dispersion medium includes a buffer.
上述技术方案中，所述杂交瘤细胞株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏号为CGMCC No.14284。In the above technical scheme, the hybridoma cell line is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 14284.
本发明还公开了一种检测DLL4蛋白的表达水平的试剂或者分选DLL4+DC的试剂的制备方法，在动物腹腔内接种杂交瘤细胞株，将动物腹水液分离、纯化制备单克隆抗体；将所述单克隆抗体与分散介质混合制备得到检测DLL4蛋白的表达水平的试剂或者分选DLL4+DC的试剂。The invention also discloses a preparation method of a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+DC, inoculating a hybridoma cell strain in the abdominal cavity of an animal, separating and purifying the ascitic fluid of the animal to prepare a monoclonal antibody; The monoclonal antibody is mixed with the dispersion medium to prepare a reagent for detecting the expression level of DLL4 protein or a reagent for sorting DLL4+DC.
上述技术方案中，所述分散介质包括缓冲液。In the above technical solution, the dispersion medium includes a buffer.
上述技术方案中，所述杂交瘤细胞株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏号为CGMCC No.14284。In the above technical scheme, the hybridoma cell line is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 14284.
本发明公开的杂交瘤细胞株的制备方法包括以下步骤：The preparation method of the hybridoma cell strain disclosed by the present invention comprises the following steps:
（1）构建高表达人DLL4分子的转基因细胞：将人DLL4 CDS全长序列克隆入真核表达载体；转染仓鼠卵巢母细胞CHO细胞，通过药物和流式细胞仪筛选获得高表达DLL4分子的转基因细胞CHO/DLL4；用转基因细胞CHO/DLL4免疫BALB/C小鼠；(1) Construction of transgenic cells that highly express human DLL4 molecules: clone the full-length sequence of human DLL4 CDS into eukaryotic expression vectors; transfect hamster ovary blast CHO cells, and obtain high-expressing DLL4 molecules through drug and flow cytometry screening Transgenic cell CHO/DLL4; BALB/C mice were immunized with transgenic cell CHO/DLL4;
（2）获取融合细胞生长克隆：从免疫合格小鼠无菌取出脾脏细胞作为抗原致敏的B细胞，按照常规方法，将B细胞与骨髓瘤细胞AG8株融合，然后利用常规的融合细胞HAT筛选方法进行筛选，进而获取融合细胞生长克隆；(2) Obtain fusion cell growth clones: aseptically remove spleen cells from immune-qualified mice as antigen-sensitized B cells, and fuse B cells with myeloma cell AG8 strain according to conventional methods, and then use conventional fusion cell HAT screening method to screen, and then obtain fusion cell growth clones;
（3）应用Western Blot和流式细胞仪等生化和免疫学技术筛选和鉴定后，挑选出具有高抗体分泌水平的杂交瘤细胞株，所述杂交瘤细胞株保藏在中国微生物菌种保藏管理委员会普通微生物中心，分类命名为分泌抗人DLL4分子单克隆抗体杂交瘤细胞株6F12。(3) After screening and identification by biochemical and immunological techniques such as Western Blot and flow cytometry, a hybridoma cell line with a high antibody secretion level is selected, and the hybridoma cell line is preserved in the China Committee for the Preservation of Microorganisms General Microbiology Center, classified and named as hybridoma cell line 6F12 secreting anti-human DLL4 molecule monoclonal antibody.
上述杂交瘤细胞株的保藏信息为，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号；保藏时间：2017年6月7日；保藏号：CGMCC No.14284；分类命名：分泌小鼠抗人DLL4分子单克隆抗体杂交瘤细胞株6F12。The preservation information of the above-mentioned hybridoma cell line is, preservation unit: General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee, preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; preservation time: June 2017 7th; preservation number: CGMCC No.14284; classification and designation: hybridoma cell line 6F12 secreting mouse anti-human DLL4 monoclonal antibody.
上述技术方案中，步骤（1）中高表达人DLL4分子的转基因细胞CHO/DLL4具有较强的免疫原性，并且所表达的抗原分子的空间构型能以自然状态暴露于细胞膜表面，从而可更有效地激发机体的免疫反应。In the above technical scheme, the transgenic cell CHO/DLL4 that highly expresses human DLL4 molecule in step (1) has strong immunogenicity, and the spatial configuration of the expressed antigen molecule can be exposed on the surface of the cell membrane in a natural state, so that it can be more Effectively stimulate the body's immune response.
上述技术方案中，步骤（1）中，制备CHO/DLL4细胞的方法可以按照本领域技术人员熟知的DNA操作技术（例如参见Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbour，1989）进行基因的分离、核苷酸片段的切割与连接、克隆和表达载体的构建及扩增、核苷酸序列的分析与鉴定、细胞的转化和培养。In the above technical scheme, in step (1), the method for preparing CHO/DLL4 cells can be carried out according to DNA manipulation techniques well known to those skilled in the art (for example, refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989) Isolation of genes, cleavage and ligation of nucleotide fragments, construction and amplification of cloning and expression vectors, analysis and identification of nucleotide sequences, transformation and culture of cells.
本发明公开的由上述杂交瘤细胞株制备的单克隆抗体，为抗人DLL4单克隆抗体，命名为单克隆抗体6F12。The monoclonal antibody prepared by the hybridoma cell line disclosed in the present invention is an anti-human DLL4 monoclonal antibody, named monoclonal antibody 6F12.
本发明还提供了所述单克隆抗体6F12的重链可变区氨基酸序列：SEQ.ID.NO:1；轻链可变区氨基酸序列：SEQ.ID.NO:2。The present invention also provides the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6F12: SEQ.ID.NO:1; the amino acid sequence of the light chain variable region: SEQ.ID.NO:2.
SEQ.ID.NO:1：SEQ.ID.NO:1:
EVHVKQSGPELVKPGASVKMSCKASGYTFTSYLLHWVKQKPGQGLEWIGYIIPYNDGTRYNEKFKGKATLTSDKSSNTAYMELSSLTSEDSAVYYCAREGTGTGAFDYWGQGTSLTVSSEVHVKQSGPELVKPGASVKMSCKASGYTFTSYLLHWVKQKPGQGLEWIGYIIPYNDGTRYNEKFKGKATLTSDKSSNTAYMELSLTSEDSAVYYCAREGTGTGAFDYWGQGTSLTVSS
对应于：corresponds to:
Glu Val His Val Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser ValLys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Leu Leu His Trp ValLys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Ile Pro Tyr Asn AspGly Thr Arg Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys SerSer Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val TyrTyr Cys Ala Arg Glu Gly Thr Gly Thr Gly Ala Phe Asp Tyr Trp Gly Gln Gly ThrSer Leu Thr Val Ser SerGlu Val His Val Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser ValLys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Leu Leu His Trp ValLys Gln Lys Pro Gly Gly Gly Leu Glu Trp Ile Gly Tyr Ile Ile Pro Tyr Asn AspGly Thr Arg Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys SerSer Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val TyrTyr Cys Ala Arg Glu Gly Thr Gly Thr Gly Ala Phe Asp Tyr Trp Gly Gln Gly ThrSer Leu Thr Val Ser Ser
SEQ.ID.NO:2：SEQ.ID.NO:2:
DIVLTQSPAIMSASPGEKVTMTCRASSSVNYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPYTFGGGTKLEIKDIVLTQSPAIMSASPGEKVTMTCRASSSVNYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPYTFGGGTKLEIK
对应于：corresponds to:
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu LysVal Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln GlnLys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser GlyVal Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile SerArg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr ProTyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysAsp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu LysVal Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln GlnLys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser GlyVal Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile SerArg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr ProTyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
本发明公开的检测DLL4蛋白的表达水平的试剂或者分选DLL4+DC的试剂，由上述单克隆抗体6F12与分散介质混合制备得到；所述分散介质包括缓冲液，可以检测DLL4蛋白的表达水平或者分选DLL4+DC。The reagent for detecting the expression level of DLL4 protein or the reagent for sorting DLL4+DC disclosed in the present invention is prepared by mixing the above-mentioned monoclonal antibody 6F12 with a dispersion medium; the dispersion medium includes a buffer, which can detect the expression level of DLL4 protein or Sort DLL4+DC.
本发明与现有技术相比具有下列优点：Compared with the prior art, the present invention has the following advantages:
本发明所制备的抗人DLL4单克隆抗体6F12可识别不同的DLL4分子抗原结合位点；其对细胞上DLL4蛋白具有高效价，高特异性和高识别能力，可用于科研Western Blot检测DLL4蛋白的表达水平。并且通过体外实验发现，单克隆抗体6F12与DC的结合与商品化的克隆MHD4-46相比并不会阻断DLL4+DC诱导Naïve T cells向Th1方向分化的能力；可用于流式细胞仪分选DLL4+DC，并且不会对下一步的DLL4+DC诱导Naïve T cells向Th1方向分化的功能性实验造成影响。The anti-human DLL4 monoclonal antibody 6F12 prepared by the present invention can recognize different antigen-binding sites of DLL4 molecules; it has high titer, high specificity and high recognition ability for DLL4 protein on cells, and can be used for Western Blot detection of DLL4 protein in scientific research The expression level. And through in vitro experiments, it was found that the combination of monoclonal antibody 6F12 and DC did not block the ability of DLL4+DC to induce Naïve T cells to differentiate into Th1 direction compared with the commercial clone MHD4-46; it can be used for flow cytometry analysis DLL4+DC is selected, and it will not affect the next functional experiment of DLL4+DC inducing Naïve T cells to differentiate into Th1 direction.
附图说明Description of drawings
图1 为实施例一中以流式细胞术分析商品化抗人DLL4抗体对转基因细胞CHO/DLL4上DLL4分子的识别结果图；Figure 1 is a flow cytometric analysis of the results of recognition of DLL4 molecules on transgenic cells CHO/DLL4 by commercial anti-human DLL4 antibodies in Example 1;
图2 为实施例一中6F12杂交瘤细胞株染色体的核型分析图（放大1000倍）；Fig. 2 is the karyotype analysis diagram of the chromosome of the 6F12 hybridoma cell line in Example 1 (enlarged 1000 times);
图3 为实施例一中以Western Blot分析单克隆抗体6F12识别抗原的结果图；Fig. 3 is the result figure of analyzing the antigen recognition of monoclonal antibody 6F12 by Western Blot in embodiment 1;
图4为实施例一中以流式细胞术分析单克隆抗体6F12对转基因细胞CHO/DLL4上DLL4分子的识别结果图；Figure 4 is a flow cytometric analysis of the recognition results of the monoclonal antibody 6F12 on the DLL4 molecule on the transgenic cell CHO/DLL4 in Example 1;
图5为实施例一中以流式细胞术分析单克隆抗体6F12识别的抗原位点的竞争性抑制的结果图；Fig. 5 is a result diagram of the competitive inhibition of the antigenic site recognized by the monoclonal antibody 6F12 analyzed by flow cytometry in Example 1;
图6为实施例二中以流式细胞术分析抗人DLL4单克隆抗体6F12对DLL4阳性的mDC诱导CD4+ Naïve T cell向Th1方向分化过程的作用。Fig. 6 is the flow cytometry analysis of the effect of anti-human DLL4 monoclonal antibody 6F12 on DLL4-positive mDCs inducing the differentiation process of CD4+ Naïve T cells towards Th1 in Example 2.
具体实施方式detailed description
下面结合具体实施例来进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
实施例一 抗人DLL4单克隆抗体的制备Example 1 Preparation of anti-human DLL4 monoclonal antibody
1、转基因细胞CHO/DLL4的建立1. Establishment of transgenic cells CHO/DLL4
（1）人DLL4基因的克隆(1) Cloning of human DLL4 gene
包含有人DLL4 全长CDS片段的质粒由厦门大学韩家淮实验室惠赠。以设计的带有限制性酶切位点的引物（表1）进行PCR扩增，反应条件为94℃变性60s，55℃退火60s，72℃延伸2min，共35 个循环后，再于72℃延伸5 min，得到全长片段；PCR产物通过回收试剂盒进行纯化。The plasmid containing the full-length CDS fragment of human DLL4 was donated by Han Jiahuai Laboratory of Xiamen University. PCR amplification was carried out with the designed primers with restriction enzyme sites (Table 1). The reaction conditions were denaturation at 94°C for 60s, annealing at 55°C for 60s, and extension at 72°C for 2min. The full-length fragment was obtained by extending for 5 min; the PCR product was purified by a recovery kit.
表1 扩增引物序列
（2）人DLL4表达载体的构建(2) Construction of human DLL4 expression vector
分别用限制性内切酶BamH I和EcoR I对回收的PCR产物和表达载体pcDNA3.1进行切割，反应后的PCR产物和表达载体通过琼脂糖凝胶电泳进行分离，将含有目的条带的凝胶切下，利用回收试剂盒进行回收。在T4连接酶的作用下将PCR产物与表达载体链接，并转化感受态菌Top10。将转化后的细菌涂于含氨苄的平板上，培养过夜后挑取阳性菌落，煮菌并进行PCR鉴定，排除假阳性菌落后，保种并送测序。测序结果通过NCBI网站上Blast比对，选取序列一致无任何突变的克隆。利用质粒提取试剂盒将构建好的质粒进行提取，表达载体命名为pcDNA3.1/DLL4。The recovered PCR product and expression vector pcDNA3.1 were cut with restriction endonucleases BamH I and EcoR I respectively, and the reacted PCR product and expression vector were separated by agarose gel electrophoresis. The gel was cut off and recovered using a recovery kit. Link the PCR product with the expression vector under the action of T4 ligase, and transform the competent strain Top10. Spread the transformed bacteria on a plate containing ampicillin, pick up positive colonies after culturing overnight, boil the bacteria and carry out PCR identification, after eliminating false positive colonies, preserve the species and send them for sequencing. The sequencing results were compared by Blast on the NCBI website, and clones with consistent sequences without any mutations were selected. The constructed plasmid was extracted using a plasmid extraction kit, and the expression vector was named pcDNA3.1/DLL4.
（3）稳定表达人DLL4的CHO转基因细胞的构建(3) Construction of CHO transgenic cells stably expressing human DLL4
将表达载体pcDNA3.1/DLL4用脂质体法转染预铺于6孔板中的CHO细胞，整个过程按试剂盒Lipofectamine(TM) 3000 操作手册进行。转染过夜后更换含10%FBS的1640培养基至2ml/孔，继续培养至48h后取部分细胞利用流式细胞术检测GFP阳性率。通过GFP阳性率来判断表达载体转染的效率。同时将部分细胞按适当比例稀释后，重新铺于6孔板中，使用含600mg/L G418（通过预筛选确定适宜的G418浓度）的选择性培养基，筛选培养；待具有抗性的转基因细胞生长至足够数量，利用商品化的抗人DLL4抗体标记，通过分选流式细胞仪将高表达人DLL4的转基因CHO细胞群体分选出来。分选获得的细胞通过亚克隆，挑选出单克隆细胞株。流式细胞仪检测挑选的单克隆细胞株上人DLL4的表达水平，挑选出阳性率和表达水平最高的克隆，参见图1。The expression vector pcDNA3.1/DLL4 was transfected into CHO cells pre-plated in a 6-well plate by the liposome method, and the whole process was carried out according to the operation manual of the kit Lipofectamine(TM) 3000. After overnight transfection, the 1640 medium containing 10% FBS was replaced to 2ml/well, and after 48 hours of continuous culture, some cells were taken to detect the positive rate of GFP by flow cytometry. The efficiency of expression vector transfection was judged by the positive rate of GFP. At the same time, after diluting some cells in an appropriate ratio, they were re-plated in a 6-well plate, and were screened and cultured using a selective medium containing 600 mg/L G418 (the appropriate concentration of G418 was determined by pre-screening); the transgenic cells with resistance After growing to a sufficient number, the transgenic CHO cell population highly expressing human DLL4 was sorted out by sorting flow cytometry using a commercially available anti-human DLL4 antibody. The cells obtained by sorting are subcloned to select monoclonal cell lines. The expression level of human DLL4 on the selected monoclonal cell lines was detected by flow cytometry, and the clone with the highest positive rate and expression level was selected, as shown in Figure 1.
2、分泌特异性鼠抗人DLL4抗体的杂交瘤细胞株的制备2. Preparation of hybridoma cell lines secreting specific mouse anti-human DLL4 antibody
使用如上得到的高表达人DLL4分子的转基因细胞（CHO/DLL4）作为免疫原，三次免疫接种Balb/c小鼠（107/500ul/只）（间隔3周）。末次免疫后第四天，取小鼠脾脏细胞与P3X63Ag8小鼠骨髓瘤细胞株进行细胞融合（共10块96孔板）。以高表达人DLL4分子的CHO/DLL4为阳性对照以及CHO/mock为阴性对照，细胞按1：1的比例混合后，用间接免疫荧光法对杂交瘤培养物上清进行初步筛选。筛选出阳性阴性比例复合1：1特征的克隆。阳性克隆经复筛和亚克隆后获得稳定地分泌特异性鼠抗人DLL4抗体的杂交瘤细胞株6F12。Using the transgenic cells (CHO/DLL4) highly expressing human DLL4 molecules obtained above as the immunogen, Balb/c mice (107 /500ul/mouse) were immunized three times (interval 3 weeks). On the fourth day after the last immunization, mouse spleen cells were taken for cell fusion with P3X63Ag8 mouse myeloma cell line (10 96-well plates in total). CHO/DLL4, which highly expresses human DLL4 molecules, was used as a positive control and CHO/mock was used as a negative control. After the cells were mixed at a ratio of 1:1, the hybridoma culture supernatant was initially screened by indirect immunofluorescence. The clones with a composite 1:1 feature in the ratio of positive to negative were screened out. After rescreening and subcloning the positive clones, a hybridoma cell line 6F12 stably secreting specific mouse anti-human DLL4 antibody was obtained.
上述杂交瘤细胞株的保藏信息为，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号；保藏时间：2017年6月7日；保藏号：CGMCC No.14284；分类命名：分泌小鼠抗人DLL4分子单克隆抗体杂交瘤细胞株6F12。The preservation information of the above-mentioned hybridoma cell line is, preservation unit: General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee, preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; preservation time: June 2017 7th; preservation number: CGMCC No.14284; classification and designation: hybridoma cell line 6F12 secreting mouse anti-human DLL4 monoclonal antibody.
杂交瘤细胞经体外持续传代后，仍能稳定地分泌特异性抗体；对杂交瘤细胞株的染色体分析显示，参见图2，这两组杂交瘤细胞的染色体数目为80-110。After continuous passage in vitro, the hybridoma cells can still secrete specific antibodies stably; the chromosome analysis of the hybridoma cell lines shows that, as shown in Figure 2, the number of chromosomes of the two groups of hybridoma cells is 80-110.
3、抗人DLL4单克隆抗体的生产与特性鉴定3. Production and characterization of anti-human DLL4 monoclonal antibody
（1）采用腹水体内诱生方法生产单克隆抗体(1) Production of monoclonal antibodies by in vivo induction in ascites
取6-8周龄的雌性Balb/c小鼠，腹腔内注入Pristane （0.5ml/只）。一周后腹腔内接种杂交瘤细胞（1×106/只），同时再次腹腔内注射Pristane与福氏不完全佐剂的等体积混合物（0.2ml/只）。5-10天后收获腹水，并离心取上清于-80℃保存。Take 6-8 week old female Balb/c mice and inject Pristane (0.5ml/mouse) intraperitoneally. One week later, hybridoma cells (1×106 /monkey) were inoculated intraperitoneally, and an equal-volume mixture of Pristane and Freund's incomplete adjuvant (0.2ml/monkey) was intraperitoneally injected again. After 5-10 days, ascitic fluid was harvested, and the supernatant was collected by centrifugation and stored at -80°C.
腹水液经去除纤维蛋白和盐析处理后，以蛋白 G亲和柱层析法纯化。收集蛋白峰流出液，对磷酸盐缓冲液（PBS）透析后用751紫外分光光度计测定抗体蛋白浓度为0.8~1.8mg/ml。SDS-PAGE结果表明， 6F12分泌的鼠抗人DLL4抗体可识别人DLL4重组蛋白（参见图3）。间接免疫荧光法分析结果表明，纯化的单克隆抗体的效价在1:10000以上（参见图4）。The ascitic fluid was purified by protein G affinity column chromatography after removing fibrin and salting out. The protein peak effluent was collected, dialyzed against phosphate buffered saline (PBS), and the antibody protein concentration was determined to be 0.8-1.8 mg/ml with a 751 ultraviolet spectrophotometer. SDS-PAGE results showed that the mouse anti-human DLL4 antibody secreted by 6F12 could recognize human DLL4 recombinant protein (see Figure 3). The results of indirect immunofluorescence analysis showed that the titer of the purified monoclonal antibody was above 1:10000 (see Figure 4).
（2）Ig亚类鉴定(2) Ig subclass identification
采用试纸快速测定（Argen公司）法鉴定Ig亚类，结果显示6F12为小鼠IgG2b型抗体。The Ig subclasses were identified by rapid test paper (Argen Company), and the results showed that 6F12 was a mouse IgG2b antibody.
（3）抗体识别抗原位点的竞争性抑制试验(3) Competitive inhibition test of antibody recognition antigen site
在LPS和R848刺激活化24h后的人PBMC中（1x106/管）悬液加入单克隆抗体6F12（1：500，每管2ug），4℃孵育30分钟。洗细胞后依次加入Lin、HLA-DR、CD1C、CD123、DLL4荧光抗体，4℃孵育30分钟。再次洗涤后用流式细胞仪分析，同时设阳性和阴性对照，结果见图5。Add monoclonal antibody 6F12 (1:500, 2ug per tube) to the human PBMC (1x106 /tube) suspension after 24h stimulation and activation by LPS and R848, and incubate at 4°C for 30 minutes. After washing the cells, add Lin, HLA-DR, CD1C, CD123, DLL4 fluorescent antibodies in sequence, and incubate at 4°C for 30 minutes. After washing again, it was analyzed by flow cytometry, and positive and negative controls were set at the same time. The results are shown in Figure 5.
实施例二 单抗对DC的体外生物学效应Example 2 In vitro biological effect of monoclonal antibody on DC
本实施例描述本发明的抗人DLL4单克隆抗体对DLL4阳性的mDC诱导CD4+ Naïve Tcell向Th1方向分化过程的作用This example describes the effect of the anti-human DLL4 monoclonal antibody of the present invention on the differentiation process of DLL4-positive mDCs inducing CD4+ Naïve T cells to Th1
新鲜人外周血通过Ficoll分离获得人PBMC。使用美天妮的商品化分选试剂盒（CD1c+Dendritic Cell Isolation Kit），按美天妮提供的实验方案从PBMC中分选CD1c+ DC，获得的细胞通过流式细胞仪检测，纯度在90%以上。使用含10% FBS的RPMI-1640培养基进行培养，加入R848（终浓度为1ug/ml）和LPS（终浓度为100ng/ml）对DC刺激24h。Human PBMCs were obtained from fresh human peripheral blood by Ficoll separation. Using the commercially available sorting kit (CD1c+ Dendritic Cell Isolation Kit) of Mytini, CD1c+ DCs were sorted from PBMCs according to the protocol provided by Mytini. The obtained cells were detected by flow cytometry, and the purity was 90 %above. Cultured in RPMI-1640 medium containing 10% FBS, DC was stimulated by adding R848 (final concentration 1ug/ml) and LPS (final concentration 100ng/ml) for 24h.
第2天采用另一份新鲜人外周血通过Ficoll分离获得人PBMC。使用Stem Cell的商品化分选试剂盒（EasySep Human CD4+ T cell Isolation Kit），按Stem Cell提供的实验方案从PBMC中分选CD4+T cell，获得的细胞通过流式细胞仪检测，纯度在95%以上。使用CFSE对分选获得的细胞进行标记。标记完成后按T cell：DC为10：1的比例，铺于U型底96孔（CD4+ T cell为1x106/孔）进行混合培养。培养第4天进行补液。实验共设三组，分别为：对照组；加入6F12（2ug/孔）；加入6F12（2ug/孔）。On the second day, another fresh human peripheral blood was used to obtain human PBMCs by Ficoll separation. Using Stem Cell's commercially available sorting kit (EasySep Human CD4+ T cell Isolation Kit), CD4+ T cells were sorted from PBMC according to the experimental protocol provided by Stem Cell, and the obtained cells were detected by flow cytometry. above 95. The sorted cells were labeled with CFSE. After the labeling is completed, the ratio of T cell:DC is 10:1, spread in U-shaped bottom 96 wells (CD4+ T cell: 1x106 /well) for mixed culture. On the 4th day of culture, rehydration was carried out. Three groups were set up in the experiment, namely: control group; addition of 6F12 (2ug/well); addition of 6F12 (2ug/well).
培养7天后收集细胞，使用eBioscience的商品化固定和破膜剂，按eBioscience提供的实验方案对细胞先进行CD4的细胞膜染色，再进行IFNg细胞内染色。流式检测结果显示，与对照组相比，6F12抗体作用组的T细胞中CFSE Low群体细胞内IFNg的表达水平和比例没有明显的改变（参见图6），独立重复实验显示各组间不具有统计学差异。说明单克隆抗体6F12与DC的结合并不会阻断DLL4+ DC诱导Naïve T cells向Th1方向分化的能力。After culturing for 7 days, the cells were collected, and using eBioscience's commercial fixation and membrane breaking agent, the cells were first stained for CD4 cell membrane and then for IFNg intracellular staining according to the experimental protocol provided by eBioscience. The results of flow cytometry showed that, compared with the control group, the expression level and ratio of IFNg in the CFSE Low population of T cells in the 6F12 antibody group did not change significantly (see Figure 6). statistical difference. It shows that the combination of monoclonal antibody 6F12 and DC will not block the ability of DLL4+ DC to induce Naïve T cells to differentiate into Th1 direction.
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| CN201710677650.3ACN107383195B (en) | 2017-08-09 | 2017-08-09 | Preparation method of anti-human DLL4 monoclonal antibody 6F12 |
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| CN102741288A (en)* | 2009-08-29 | 2012-10-17 | 雅培制药有限公司 | Therapeutic dll4 binding proteins |
| CN102906113A (en)* | 2010-03-02 | 2013-01-30 | Abbvie公司 | Therapeutic DLL4-binding protein |
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| CN104428319A (en)* | 2012-07-02 | 2015-03-18 | 韩华石油化学株式会社 | Novel monoclonal antibody binding specifically to dll4 and use thereof |
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