CN102008719A - Immunomicrosphere for overcoming B cell immunological tolerance and application thereof - Google Patents

Immunomicrosphere for overcoming B cell immunological tolerance and application thereof Download PDF

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CN102008719A
CN102008719A CN2010105712771A CN201010571277A CN102008719A CN 102008719 A CN102008719 A CN 102008719A CN 2010105712771 A CN2010105712771 A CN 2010105712771A CN 201010571277 A CN201010571277 A CN 201010571277A CN 102008719 A CN102008719 A CN 102008719A
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microspheres
cell
immune
protein
cells
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CN2010105712771A
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张尚权
谈立松
陈宇光
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上海微球生物科技有限公司
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Abstract

The invention relates to an immunomicrosphere for overcoming B cell immunological tolerance and application thereof. The immunomicrosphere comprises a microsphere medium, wherein the outside of the microsphere medium is coated with vaccine antigen, and the inside of the microsphere medium is coated with immune carrier protein for activating T cells. The invention also relates to application of the immunomicrosphere for overcoming the B cell immunological tolerance in preparing vaccines and immunological adjuvants. By using the technical scheme, the immunomicrosphere is combined with B cell receptor through the antigen and phagocytized by B cells, and the carrier protein is released and degraded in the cells; the immunomicrosphere is presented to Th cells through B cell MHC II to activate the TH cells to release cell factors, thereby promoting the activation and proliferation of the B cells; and high-titer antibody is generated through the affinity maturation process. The antigen protein and the immune carrier protein are respectively arranged on the surface of the microsphere and inside the microsphere, thereby preventing the antigen protein epitope and the Th cell activation epitope from competing with the B cell receptor.

Description

克服B细胞免疫耐受的免疫微球及其应用 Immune microspheres B cell tolerance to overcome its application

技术领域 FIELD

[0001] 本发明涉及免疫领域,特别涉及一种克服B细胞免疫耐受的免疫微球及其应用。 [0001] The present invention relates to the field of immunization, particularly to a B cell tolerance against immune microsphere and its application.

背景技术 Background technique

[0002] 抗原(Ag)是指能与T、B淋巴细胞的TCR或BCR结合,促使其增殖、分化,产生抗体或致敏淋巴细胞,进而发挥免疫效应的物质。 [0002] The antigen (Ag) can refer, B lymphocytes in combination with a TCR or BCR T, which promote proliferation, differentiation, produce antibodies or sensitized lymphocytes, then play an immune effects. 抗原的分类方法很多,根据诱生抗体时需否Th细胞参与,可分为胸腺依赖性抗原(TD-Ag)和胸腺非依赖性抗原(TI-Ag)。 Classification of many antigens, antibodies according to the time required to induce Th cells involved NO can be divided into thymus-dependent antigen (TD-Ag) and thymus-independent antigens (TI-Ag). 绝大多数蛋白质抗原,如病原微生物、血细胞、血清蛋白等均属TD-Ag。 The vast majority of protein antigen, such as pathogenic microorganisms, blood cells, serum protein, etc. are TD-Ag. 胸腺依赖性抗原诱导的抗体产生过程需要T细胞的参与。 Thymus-dependent antigen-induced antibody production process requires involvement of T cells. 抗体产生的指令来自两种信号:抗原与B细胞表面抗原受体结合和辅助性T淋巴细胞(Th)的辅助刺激信号。 The antibodies produced from two instruction signal: B surface antigen and secondary antigen receptor cell binding and T helper (Th) of the stimulation signal.

[0003] 抗原蛋白表位与B细胞膜受体结合并发生内化,是第一信号。 [0003] The antigenic epitopes and B cell membrane receptors binding and internalization occurs, is the first signal. B细胞吞噬免疫微球后在B细胞内体(endosome)或溶酶体(Iysome)中释放抗原并被蛋白酶水解,其中合适的片断与细胞MHCII结合提呈给Th细胞。 After immunization B cells engulf the microspheres within the B cells (endosome) or lysosomes (Iysome) and proteolytic release of antigen, wherein the fragment a suitable cell binding MHCII presentation to Th cells. 实现Th细胞活化并释放细胞因子作为第二信号促使B细胞转化成分泌抗体的浆细胞。 Th cell activation and achieve the release of cytokines as a second signal causes B cells into antibody-secreting plasma cells.

[0004] 接种疫苗是人类抵御和控制传染性疾病的有效手段之一。 [0004] Vaccination is an effective means against humanity and control of infectious diseases. 传统意义上的疫苗主要是预防性疫苗,其应用对象是健康群体的易感动物及人群,其疫苗成分一般均为微生物保护性抗原,可在健康肌体中激活特异性免疫应答,产生特异性抗体和细胞毒T淋巴细胞(CTL),从而获得对该病原体的免疫预防能力,但对已发病的个体不能产生有效的免疫应答。 Vaccine in the traditional sense essentially preventive vaccine application object is susceptible animal population health and population, its vaccine components are generally microbial protective antigen, can activate specific immune response in healthy organism, the production of specific antibodies and cytotoxic T lymphocytes (the CTL), the ability to obtain immunization pathogens, but can not produce disease subject has an effective immune response.

[0005] 治疗性疫苗(Therapeutic Vaccine)是近年建立和发展起来的免疫治疗新概念,是 [0005] therapeutic vaccine (Therapeutic Vaccine) in recent years established and developed the new concept of immunotherapy, is

指能够打破慢性感染者体内免疫耐受,重建或增强免疫应答的疫苗。 It refers to the ability to break immune tolerance to chronic infection, rebuild or enhance the immune response to the vaccine. 治疗性疫苗能在已患病个体诱导特异性免疫应答,消除病原体或异常细胞,使疾病得以治疗,是抗病毒、 抗肿瘤的新治疗手段。 Therapeutic vaccines can induce specific immune responses have been affected individuals, eliminate pathogens or abnormal cells, the disease is treated, is anti-viral, anti-cancer new treatment.

[0006] 肿瘤是治疗疫苗重点研究领域。 [0006] cancer treatment vaccine research priority areas. 用于制备疫苗的抗原有生长因子(EGF、TGF、 VEGF)及相应受体(EGFR、Her2、VEGFR)肿瘤相关抗原CEA、MUCl等用于实体肿瘤治疗。 For the preparation of a vaccine antigen are growth factors (EGF, TGF, VEGF) and their receptors (EGFR, Her2, VEGFR) of tumor associated antigen CEA, MUCl and other for the treatment of solid tumors. CD20用于B细胞淋巴病的疫苗。 CD20 B lymphoid vaccine for disease cells. Idiotype是另一类用于B细胞和T细胞淋巴细胞白血病。 Idiotype is another for B cells and T lymphocytic leukemia cell.

[0007] 默克制药公司目前正对一种用于非小细胞肺癌(最常见的肺癌种类)治疗的疫苗进行第三期临床试验,试验计划于2010年结束。 [0007] Vaccines Merck is currently on for non-small cell lung cancer (the most common type of lung cancer) treatment of the Phase III clinical trial, trial is scheduled to end in 2010. 该疫苗被设计用于诱发癌细胞表面的MUCl分子产生一种免疫反应。 The vaccine is designed to induce cell surface molecules MUCl produce an immune response. 1300名参与此项试验的患者遍布全球,其中包括巴西、 中国、印度和墨西哥这些发展中国家。 1300 patients participating in this trial around the world, including Brazil, China, India and Mexico, these developing countries. 这一大范围的受试者征集反映肺癌是一个全球性的问题。 This collection reflects a wide range of subjects Lung cancer is a global problem. 全球首个癌症治疗性EGF疫苗在古巴获准上市用于肺癌治疗。 The world's first therapeutic cancer vaccine in Cuba EGF approved for marketing for the treatment of lung cancer.

[0008] 瑞士医药集团塞托斯生物技术公司和英国Protherics公司两家都在对高血压疫苗进行二期临床试验。 [0008] Swiss pharmaceutical group Setuo Si biotechnology companies and two companies in the UK Protherics hypertension vaccine Phase II clinical trials. 是以血管紧张素为疫苗,产生封闭抗体。 Angiotensin is a vaccine, produce blocking antibodies.

[0009] 法国Neovacs公司Zagury等将人肿瘤坏死因子连接一种免疫促进剂——钥孔虫戚血蓝蛋白(KLH)。 [0009] French company Neovacs Zagury et al. Tumor Necrosis Factor connected to an immune enhancers - keyhole limpet hemocyanin (KLH). 用此复合物免疫接种转基因小鼠,可完全保护小鼠免于重复注射肿瘤坏死因子的伤害,用于类风湿疾病治疗。 With this composite immunized transgenic mice can be completely protected mice from harm repeated injection of tumor necrosis factor for the treatment of rheumatoid diseases. 拟用以代替昂贵的单克隆抗体和受体治疗用途。 Intended to replace the expensive therapeutic uses of monoclonal antibodies and receptors. 针对IgE的疫苗用于过敏性疾病、可能突现技术突破。 Vaccine against IgE for allergic diseases, may emergent technological breakthroughs. TGF用于肝硬化及动脉高压研究。 Cirrhosis and high-pressure arterial TGF for research. 治疗性疫苗极有可能复制甚至超越单克隆抗体过去10年的辉煌。 Therapeutic vaccine is likely to replicate or exceed the monoclonal antibodies over the past 10 years of glory.

[0010] 以上的疫苗均以人体内自身蛋白为抗原,产生抗体为目标,通过中和反应封闭特定分子的功能,或通过抗体的ADCC效应和补体杀伤效应实现治疗目的 [0010] Vaccines are more self-protein in humans as an antigen to produce antibodies to the target, and by the reaction of a particular molecule sealing function, object, or to achieve a therapeutic effect by ADCC and complement killing effects of antibodies

[0011] 由上可知,目前的治疗性疫苗所采用的抗原通常选用人体或者动物的自身蛋白,或称内源性蛋白。 [0011] From the above, current therapeutic vaccine antigen used in human or animal usually use self-protein, also known as endogenous proteins.

[0012] 内源性蛋白不能诱导动物或人产生高滴度抗体。 [0012] The endogenous proteins inducing an animal or human can not produce high antibody titers. 称之谓B细胞免疫耐受。 Commonly known that B cell tolerance.

[0013] 实际上但是针对自身蛋白反应的B细胞还是会少量产生。 [0013] However, actually self-proteins against B cell response will still produce a small amount. 在二级淋巴细胞组织中因体细胞突变机制使B细胞V区基因突变。 In the two mutant lymphocytes in the mechanism due to somatic mutations in a gene B cell V region. 不能产生能针对自身蛋白抗体的机理在于辅助T细胞免疫耐受。 That can not produce helper T cell tolerance against the self-protein antibody of the mechanism.

[0014] 目前认为维持体内自身免疫耐受状态的主要机制是在中枢免疫器官发生的T细胞克隆清除,在外周中诱导自身潜能细胞进入免疫无能(anergy)状态,及部分淋巴细胞对自身蛋白的免疫忽视(ignorance)。 [0014] presently considered to be the main mechanism for maintaining the state of self-tolerance in vivo clonal deletion of T cells is the central immune organ occurs, the outer peripheral cells into their potential to induce anergy (anergy) state, and some self-proteins lymphocytes immune ignored (ignorance). 内源性蛋白的表位可与B细胞结合,但不能诱导B 细胞产生抗体 Epitope endogenous proteins may be combined with B cells, but unable to induce B cells to produce antibodies

[0015] 现有的技术是与外源性强抗原蛋白,免疫学上称之谓免疫载体(immunocarrier) 的蛋白化学偶联,引入外源性蛋白用于活化Th细胞。 [0015] The prior art is highly antigenic exogenous protein, known that the immunogenic carrier (immunocarrier) chemically coupled protein immunologically, exogenous proteins introduced for Th cell activation. 经载体蛋白活化Th细胞,才可诱导B细胞产生抗体。 Carrier protein activated by Th cells, induce B cells to produce antibodies before. 在免疫应答中,B细胞识别内源性蛋白抗原表位,并提呈载体蛋白表位给CD4+Th细胞,Th细胞识别载体蛋白表位,这样载体就可把特异TB细胞连接起来(TB桥联),T细胞才能激活B细胞。 In the immune response, the B cell derived protein epitopes, and presenting an epitope to a carrier protein CD4 + Th cells, Th cells recognize epitopes of the carrier protein, so that the carrier can be connected to a specific cell TB (TB bridge Union), T cells can activate B cells.

[0016] 除了将内源性蛋白与免疫载体(immonocarrier)蛋白化学交联方法,还用基因工程技术构建含有免疫载体的融合蛋白作为异源分子。 [0016] In addition to endogenous proteins and immunogenic carrier (immonocarrier) protein chemical crosslinking method, further comprising an immunization vector construct fusion proteins by genetic engineering techniques, as a heterologous molecule.

[0017] 由此带来了新的问题:由于针对内源蛋白的B细胞在发育过程中克隆清除,与外源载体蛋白表位结合的B细胞的丰度比与内源性蛋白表位结合的B细胞的丰度高几个数量级,从而存在交联蛋白(或融合蛋白)分子内蛋白表位免疫竞争的问题。 [0017] The resulting new problems: Since for B-cell clonal deletion of endogenous protein during development, the abundance of B-cells in combination with exogenous ratio of carrier protein epitope binding with endogenous epitopes several orders of magnitude higher abundance of B-cells, there is a problem crosslinked protein (or fusion protein) epitope of a protein molecule immunized competition. 当疫苗进入人体或动物体内,外源载体蛋白表位具有竞争优势,B细胞受体有更高机率结合外源载体蛋白表位并吞噬,产生针对外源载体蛋白的高滴度抗体。 Where the vaccine into the human or animal body, the foreign carrier protein competitive epitopes, B cell receptor has a higher probability of binding a foreign carrier protein epitopes and swallowed, produce high titers of antibodies against the foreign carrier protein. 而内源性蛋白的相应抗体受抑制,只能产生低滴度抗体,很难实现临床治疗价值。 The corresponding endogenous protein antibody inhibited, can only produce low antibody titers, it is difficult to achieve clinical value.

[0018] 本发明与文献中报道的免疫微球区别在于:已有的免疫微球是将抗原吸附或化学交联于微球表面或将抗原包裹于微球内部。 [0018] Immune microspheres present invention differs from that reported in the literature: an existing immune microspheres antigen adsorbed or chemically crosslinked to the interior surface of the microspheres in the microsphere antigen or package. 功能是缓释作用或促进巨噬细胞吞噬。 Function is to slow release or promote macrophage phagocytosis. 不能起到克服B细胞免疫耐受,也不适用于内源性蛋白。 Can not play against the B cell tolerance, does not apply to an endogenous protein.

发明内容 SUMMARY

[0019] 为了解决上述问题,本发明提供一种的免疫微球,从而克服现有技术中化学交联蛋白或融合蛋白分子内表位的免疫竞争问题。 [0019] In order to solve the above problems, the present invention provides an immune microspheres, thus overcoming the prior art chemical crosslinking or fusion protein molecules within an immune competition epitope.

[0020] 一种克服B细胞免疫耐受的免疫微球,其包括微球介质,所述微球介质外部包覆有疫苗抗原,所述微球介质内部包裹有用于活化T细胞的免疫载体蛋白。 [0020] B cell tolerance overcomes immune microspheres, the microspheres comprising a medium, the medium outside the microspheres is coated with a vaccine antigen, the microspheres internal wrapping medium having immunogenic carrier protein for T cell activation .

[0021] 所述疫苗抗原的B细胞表位位于所述免疫微球的表面,所述免疫载体蛋白的T细胞表位位于所述免疫微球的内部,从而实现B细胞表位与T细胞表位的物理隔离。 [0021] The B cell epitope vaccine antigens located on the surface of the microspheres immunization, the immunogenic carrier protein T-cell epitopes located within the immune microspheres to achieve B cell epitopes and T cell epitopes bit physical isolation.

[0022] 所述的免疫载体蛋白选自:嘁血蛋白、牛血清白蛋白、卵白蛋白、基因工程表达的破伤风病毒蛋白片断、乙肝病毒壳蛋白、脑膜炎球菌蛋白、HIV外壳蛋白、感冒病毒蛋白。 Immunogenic carrier protein [0022] selected according to: limpet albumin, bovine serum albumin, ovalbumin, tetanus viral protein expression genetically engineered fragments, hepatitis B virus coat protein, Meningococcal protein, HIV coat protein, influenza virus protein.

[0023] 所述的疫苗抗原选自:表皮生长因子、血管生长因子、血管生长因子受体、 IgE,表皮生长因子受体EGFR及突变体、表皮生长因子受体肿瘤坏死因子、转化生长因子血管紧张素、CD20。 Vaccine antigens [0023] selected according to: epidermal growth factor, vascular endothelial growth factor, vascular endothelial growth factor receptor, IgE, and epidermal growth factor receptor (EGFR) mutant epidermal growth factor receptor tumor necrosis factor, transforming growth factor angiogenesis angiotensin, CD20.

[0024] 所述微球介质选自:海藻酸钙、聚乳酸、聚乳酸-乙醇酸、琼脂糖、葡聚糖。 [0024] The microspheres medium is selected from: calcium alginate, polylactic acid, polylactic acid - glycolic acid, agarose, dextran.

[0025] 所述疫苗抗原通过常规化学方法与所述微球介质偶联。 [0025] The conventional chemical methods vaccine antigen with the microspheres through the coupling medium.

[0026] 利用双功能交联试剂实现氨基-氨基、羧基-氨基、氨基-锍基的偶联。 [0026] The use of a bifunctional crosslinking reagent to achieve amino - amino group, a carboxyl group - amino, - coupling sulfonium group.

[0027] 利用戊二醛试剂实现氨基与氨基的偶联。 [0027] achieved by coupling amino group with amino reagent glutaraldehyde.

[0028] 所述的克服B细胞免疫耐受的免疫微球在疫苗制备上的应用。 [0028] Application of the B cell tolerance by the immune microspheres overcome the preparation of a vaccine.

[0029] 所述的克服B细胞免疫耐受的免疫微球在免疫佐剂上的应用。 [0029] Application of the B cell tolerance by the immune microspheres immunoadjuvant overcome.

[0030] 通过上述技术方案,本发明提供的免疫微球通过抗原与B细受体结全被B细胞吞噬,载体蛋白在细胞内释放、降解;并通过B细胞MHCII提呈给Th细胞,激活Th细胞释放细胞因子,从而促使B细胞活化、增殖;经过亲和力成熟过程产生高滴度抗体。 [0030] Through the above technical solution, the present invention provides an immune antigen and the microspheres B cells by receptor binding were all B phagocytosis, intracellular release carrier protein degradation; presentation to Th cells and B cells by MHC-II, activated Th cell cytokine release, thereby causing B cell activation, proliferation; through the process of affinity maturation to produce high antibody titers. 通过将抗原蛋白及免疫载体蛋白分置于微球表面及内部,避免了抗原蛋白表位与Th细胞活化表位同B细胞受体的竞争。 By immunogenic carrier protein and the antigen protein fraction microspheres placed on the surface and inside, to avoid antigenic epitopes and Th cell epitopes with activated B cell receptor competition.

[0031] 根据本发明的一个优选实施例,提供一种表皮生长因子(EGF)构建的微球。 [0031] According to a preferred embodiment of the present invention, there is provided an epidermal growth factor (EGF) constructed microspheres.

[0032] EGF疫苗,这种具有靶向性质的新药早已引起世界各国医药界的关注。 [0032] EGF vaccine, targeted nature of this new drug has already caused concern in the world medical community of nations. 该疫苗由古巴分子免疫学中心研发,临床研究证实用于治疗非小细胞肺癌(NSCLC)治疗有效。 The vaccine developed by the Cuban Molecular Immunology Center, clinical studies for the treatment of non-small cell lung cancer (NSCLC) treatment is effective.

[0033] 该研究成果10年前转让给美国cancerVax公司进行临床研究。 [0033] The transfer of research results to the United States 10 years ago, the company cancerVax clinical studies. 美国已经通过药品的临床试验,预计将在未来2到3年内投入应用。 The United States has drugs through clinical trials, expected to be operational in the next 2-3 years. ASCO(美国临床肿瘤学)大会连续几年均报道了EGF疫苗临床研究进展报告。 ASCO (American Society of Clinical Oncology) General Assembly for several years have reported progress report EGF vaccine clinical research. 晚期不可切除的非小细胞肺癌患者在接受一线化疗后,随机分为两组,一组接种EGF疫苗,另一组接受最佳支持治疗,接种时间为第0、7、14、21、51天,之后每月接种1次。 Advanced unresectable non-small cell lung cancer patients after receiving first-line chemotherapy, were randomly divided into two groups, one group received EGF vaccine, another group received best supportive care, time for the first vaccination day 0,7,14,21,51 after inoculation once a month. 研究结果显示:试验组患者总生存期比对照组患者提高4〜5个月,患者身体素质显著改善,抵抗力增强。 The results showed: patients in the test group to improve the overall survival of 4 to 5 months longer than patients in the control group, patients significantly improved physical fitness, enhanced resistance. 开发治疗性疫苗研究将具有重大的理论意义,拥有巨大的经济及社会效益。 Therapeutic vaccine development will be of great theoretical significance, it has a huge economic and social benefits.

[0034] 目前,秘鲁正在进行对药品的审核,预计年底能够上市,在中国,由北京百泰生物药业有限公司负责该药临床研究及产业化生产,并在2008年底在中国开展大规模临床研究,进一步验证该药对非小细胞肺癌患者的疗效。 [0034] Currently, Peru ongoing review of drugs, is expected to end can be listed in China, the Beijing Baitai Biological Pharmaceutical Co., Ltd. is responsible for clinical drug research and industrial production, and at the end of 2008 to carry out large-scale clinical in China studies to further validate the efficacy of the drug in patients with non-small cell lung cancer.

[0035] 肿瘤细胞区别于正常细胞主要特征之一是失控性生长,肿瘤细胞本身可以生成某些生长因子促进细胞无限制生长,EGF (表皮生长因子)是近年发现的在细胞增殖和分化中起重要作用的分子。 [0035] One of the main features of the tumor cells to normal cells is different from the uncontrolled growth of the tumor cells themselves can produce certain growth factors to promote unrestricted cell growth of EGF (epidermal growth factor) was recently discovered in cell proliferation and differentiation from molecular important role. EGF疫苗正是运用特异性主动免疫的方法,以肿瘤组织高表达的EGF-EGFR(表皮生长因子受体)系统信号通路为作用靶点。 EGF vaccine is the use of specific active immunotherapy approach to tumor tissue expression of EGF-EGFR (epidermal growth factor receptor) system signal path for the targets. 患者接种该疫苗后,刺激免疫系统产生针对EGF的抗体,阻断由EGF-EGFR介导的下游信号传导通路,从而诱导肿瘤细胞凋亡,为肿瘤分子靶向治疗提供了更新的选择。 Patients received the vaccine, to stimulate the immune system to produce antibodies against the EGF blocking mediated by the EGF-EGFR downstream signaling pathway, to thereby induce tumor cell apoptosis, provides the option of updating molecular targeted therapy for cancer.

[0036] 在本发明的优选实施例中,用海藻酸钙为材料制备微球,微球内包裹有牛血清白蛋白(BSA)作为活化T细胞的免疫载体蛋白(Immunocarrier)。 [0036] In a preferred embodiment of the present invention, by preparing microspheres of calcium alginate material, the microspheres wrapped with bovine serum albumin (BSA) as a carrier of activated T cells of the immune protein (Immunocarrier). 表面复以壳聚糖提供连按抗原的氨基。 Chitosan complex surface antigens provide even by amino. 以小鼠内源的表皮生长因子(mEGF)为疫苗抗原,通过化学方法偶联于微球表面。 In the endogenous mouse epidermal growth factor (of mEGF) as a vaccine antigen, coupled to the microsphere surface by chemical means. 免疫微球经尾静脉注射免疫小鼠,4周后取血常规ELISA方法测定EGF抗体滴度。 Immune microsphere injection via the tail vein of mice immunized was determined EGF antibody titers by ELISA After 4 weeks blood. 结果显示:EGF抗体滴度大于8000 ;常规方法皮下免疫,EGF滴度小于200。 The results showed that: EGF antibody titers greater than 8000; subcutaneously immunized conventional method, EGF titers less than 200.

[0037] 从而证明未经化学交联的小鼠自身蛋白可以成为完全抗原免疫获得高滴度抗体。 [0037] The mice thus demonstrating without chemical cross-linking can be completely self-protein antigen to obtain a high titer antibody. 根据本发明的一个优选实施例,本发明提供的表皮生长因子(EGF)构建的微球为EGF-微球疫苗治疗肿瘤提供实验基础。 According to a preferred embodiment of the present invention, the present invention provides constructs epidermal growth factor (EGF) microspheres experimental basis for the treatment of a tumor vaccine EGF- microspheres.

[0038] 根据本发明的一个优选实施例,提供一种血管生长因子(VEGF)构建的微球。 [0038] According to a preferred embodiment of the present invention, there is provided a vascular endothelial growth factor (VEGF) Construction of microspheres.

[0039] 肿瘤血管形成在肿瘤生长、侵袭和转移中具有重要意义,如果没有新生血管进入肿瘤为其带来足够的氧气和营养,并运走代谢产物,肿瘤直径将很少超过2〜3_, 因为仅靠周围的血管对肿瘤弥散作用是完全不够的,肿瘤只能处于休眠状态或发生退化。 [0039] Angiogenesis is important in tumor growth, invasion and metastasis, if there is no new blood vessels into the tumor for which bring in enough oxygen and nutrients, and carried away metabolites, tumor diameter will be less than 2~3_, because the blood vessels around the tumor alone is totally inadequate dispersion effect, the tumor can only occur in a dormant state or degradation. 如果血管一旦长入肿瘤组织,提供给肿瘤组织营养和氧气的方式由弥散转变成血流灌注,其代谢产物将能够被及时和彻底地清除,则肿瘤呈指数生长,显现出无限制扩张的趋势。 If the tumor tissue ingrowth once the vessels, to provide nutrients and oxygen to the tumor tissue from the diffusion mode change to the perfusion, a metabolite capable of being promptly and thoroughly removed, then the exponential growth of the tumor, showing the trend of unlimited expansion . 肿瘤的生长和血管生成是相辅相成的。 Tumor growth and angiogenesis are complementary. 肿瘤细胞产生的促血管生成因子能够刺激内皮细胞的生长及生存,而血管的生成不仅是提供营养、氧气和运走代谢物,血管内皮细胞还可向肿瘤提供生长启动的因子。 Pro-angiogenic growth factor produced by tumor cells capable of stimulating growth and survival of endothelial cells, and angiogenesis is not only provide nutrition, oxygen metabolites, and carried away, vascular endothelial cell growth factor may also be provided to the tumor started. VEGF(血管内皮生长因子)是得到最好定性的肿瘤血管形成诱发因子,阻断VEGF的功能已经成为癌症治疗中的一个重要工具。 VEGF (vascular endothelial growth factor) is the best qualitative factor-induced angiogenesis, blocking VEGF function has become an important tool in cancer therapy.

[0040] 在本发明的优选实施例中,以血管内皮细生长因子(mVEGF)为抗原,以聚乳酸-乙醇酸(PLGA)制备免疫微球,以鸡卵白蛋白为免疫载体蛋白。 [0040] In a preferred embodiment of the present invention, vascular endothelial growth factor (mVEGF,) as antigen, polylactic acid - glycolic acid (PLGA) microspheres were prepared immunization, immune chicken ovalbumin carrier protein. 微球复以壳聚糖提供偶联氨基。 Chitosan microspheres to provide a complex conjugate amino group. 戊二醛方法偶联VEGF。 Glutaraldehyde coupling method VEGF. 免疫条件与EGF微球相同。 Immune conditions and EGF microspheres same. 6周后取血常规ELISA 方法测定mVEGF抗体滴度。 Determination of antibody titer mVEGF by ELISA 6 weeks after taking blood. 结果显示,mVEGF抗体滴度达到64000。 The results showed that, mVEGF antibody titer 64000. 获得较海藻酸钙微球更好的结果。 Calcium alginate microspheres to obtain more and better results. 因此,本发明的微球为肿瘤血管抑制提供了免疫制剂。 Thus, the microspheres of the present invention provides a formulation for the immunization of the antiangiogenic.

[0041] 根据本发明的一个优选实施例,提供一种小鼠IgE C ε 3多肽构建的微球。 [0041] According to a preferred embodiment of the present invention, there is provided a mouse IgE C ε 3 polypeptide construct microspheres. IgE 介导性疾病是一类由IgE引发的变态反应性疾病的统称,因其易感者在接触环境中变应原时往往产生过多的IgE抗体,通常伴有一种以上的变态反应性疾病而得名。 IgE-mediated disorder is a type collectively IgE-induced allergic diseases, because it tends to be susceptible becomes excessive IgE antibodies original at exposure environment, usually accompanied by one or more allergic diseases named. 当变应原初次进入机体后,选择性诱导特异性B细胞产生IgE抗体应答,而血清中游离的IgE可在不结合抗原的情况下通过其Fc片段与肥大细胞和嗜碱性粒细胞表面受体结合使机体处于致敏状态。 When the initial allergen into the body, selectively inducing specific B cells to produce IgE antibody response in serum free IgE Fc fragments which may be affected by the surface of mast cells and basophils without bind antigen so that the body is binding sensitization. 机体再次接触变应原后,多价变应原与致敏细胞表面两个或者两个以上相邻的IgE抗体结合,使细胞膜表面受体发生交联,触发致敏细胞脱颗粒,合成及释放生物活性介质,从而引起局部或者全身过敏反应。 Body after re-exposure to the allergen, a multivalent allergen sensitization in combination with two or more cell surface adjacent IgE antibodies, cell membrane receptors surface crosslinking occurs, the trigger sensitized cell degranulation, synthesis and release biologically active medium, causing a localized or systemic anaphylaxis. IgE介导性疾病通常包括特应性皮炎(Atopic Dermatitis, AD)、变应性鼻炎(Allergic Rhinitis,AR)、支气管哮喘(Asthma,AS)等, 症状重者可导致过敏性休克,危及生命。 IgE mediated disorders includes atopic dermatitis generally (Atopic Dermatitis, AD), allergic rhinitis (Allergic Rhinitis, AR), bronchial asthma (Asthma, AS) and the like, can lead to severe symptoms of anaphylactic shock, a life-threatening. 变态反应性疾病发病率逐年提高趋势,已严重干扰了患者的工作和生活,由此带来的社会负担也是相当可观的。 The incidence of allergic diseases increasing year by year trend, has seriously interfered with the patient's work and life, and the resulting social burden is considerable. 由于IgE在变态反应性疾病的发生发展环节中的重要作用,目前已经成为抗变态反应性疾病治疗的一个新的靶点和研究热点。 Because of the important role of IgE in the development aspects of allergic diseases, it has become a target for new research focus and anti-allergic treatment of disease. IgE的分子结构已清楚,在IgE抗体分子与胞大细胞受体结合的分子表位,位于抗体分子Fc的C ε 3结构域。 The molecular structure of IgE clearly, the epitope molecule IgE antibody binding to the extracellular molecules large cell receptor, an antibody molecule located at the C ε 3 Fc domains. 虽然针对此区域的单克隆抗体已经FDA批准上市并在临床应用中显示出了良好的效果,但是,造价的昂贵和使用上的不便限制了其广泛应用,因此,由被动地接受这种抗体蛋白转向寻求针对该蛋白的主动免疫疫苗有着更好的应用前景。 Although monoclonal antibodies directed against this region has FDA clearance to market and show good results in clinical applications, however, the cost of expensive and inconvenient restrictions on the use of its wide application, therefore, the passively accept such an antibody protein It has turned to seek a better prospect for the active immunization of the vaccine protein. 研究构建能够诱导出针对IgE受体结合位点(C ε 3结构域)特异性中和抗体的治疗性疫苗,通过抗原抗体封闭IgE Fc片段上与IgE Fc受体结合的部位,进而阻断由IgE介导的变态反应性疾病效应途径,为IgE介导性疾病的治疗提供新的途径。 Construction capable of inducing a neutralizing antibody specific therapeutic vaccine against IgE receptor binding site (C ε 3 domains), closed on the Fc fragment of IgE binding to the Fc receptor site of IgE antibodies by an antigen, and further blocked by the ill effects of IgE-mediated allergy ways, providing new avenues for the treatment of IgE-mediated diseases.

[0042] 在本发明的优选实施例中,用化学合成方法合成了小鼠IgEFc的C ε 3结构域特异的多肽。 [0042] In a preferred embodiment of the present invention, synthesized by chemical synthesis of mouse C ε 3 IgEFc domain specific polypeptide. 序列为:GYGYQCIVDHPDFPKPIVRSITKTPGQR。 Sequence: GYGYQCIVDHPDFPKPIVRSITKTPGQR. 该多肽相应单克隆抗体 The corresponding polypeptide monoclonal antibodies

被证实能阻断IgE与肥大细胞受体结合并对过敏性疾病治疗有效。 It has been shown to block the binding of IgE to mast cell receptors and effective treatment of allergic diseases. 微球材料为PLGA/壳聚糖,包裹鸡卵白蛋白作为免疫载体。 Microsphere material is PLGA / chitosan, wrapped as ovalbumin immunogenic carrier. 戊二醛方法将多肽偶联微球。 The method of glutaraldehyde polypeptide coupled microspheres. 按实例1的方法免疫小鼠和测定抗体。 The method according to Example 1 of the immunized mice and assayed for antibody.

[0043] 结果显示:6周后小鼠IgE C ε 3结构域多肽抗体滴度大于32000。 [0043] The results show: After 6 weeks mice IgE C ε 3 domain polypeptide antibody titer greater than 32,000. 优于目前常用即与BSA化学交联常规方案。 I.e., better than conventional schemes currently used chemical crosslinking with BSA. 因此,本发明的微球为过敏性疾病提供了免疫治疗的基石出。 Thus, the microspheres of the present invention provides a basis for immunotherapy of the disease is allergic.

[0044] 根据本发明的一个优选实施例,提供一种表皮生长因子受体III型突变体(EGFRvIII)构建的微球。 [0044] According to a preferred embodiment of the present invention, there is provided an epidermal growth factor receptor type III mutant (EGFRvlll) Construction of microspheres. 表皮生长因子受体III突变体(EGFRvIII)是EGFR最常见的一种缺失型突变体。 III EGFR mutant (EGFRvlll) is the most common type EGFR deletion mutants. 相对于野生型EGFR蛋白而言,EGFRvIII在其胞外段缺失了256个氨基酸从而失去了和配体结合的功能,但是在不和配体结合的情况下其胞内段也可以发生组成性磷酸化并激活下游信号通路。 Relative to the wild-type EGFR protein, the extracellular segment EGFRvlll deletion of 256 amino acids, and thereby losing the function of ligand binding, but which constitutively phosphorylated intracellular domain may also occur without ligand binding and and activates downstream signaling pathways. 研究表明,EGFRvIII仅在肿瘤细胞上表达,如脑胶质瘤,乳腺癌,卵巢癌,前列腺癌等,在正常细胞检测不到EGFRvIII的存在。 Studies have shown that, EGFRvIII is only expressed in the tumor cells, such as gliomas, breast cancer, ovarian cancer, prostate cancer and the like, can not detect the presence of EGFRvIII in normal cells. 体外体内实验证明EGFRvIII的表达可以促进细胞转化,肿瘤细胞增殖和侵袭,并与肿瘤病人的预后密切相关。 Expression of EGFRvIII in vivo and in vitro experiments show that promote cell transformation, tumor cell proliferation and invasion, and is closely related to the prognosis of cancer patients. 因此EGFRvIII是一个肿瘤诊断及治疗的理想靶点。 EGFRvIII is therefore an ideal target for a cancer diagnosis and treatment.

[0045] 针对EGFRvIII的抗体可被应用于临床治疗,由于正常组织不表达EGFRvIII,所以EGFRvIII抗体的特异性更强。 [0045] against EGFRvIII antibodies it can be used in clinical treatment, due to normal tissues do not express EGFRvIII, so EGFRvIII-specific antibodies stronger. YlO是特异针对人EGFRvIII的一种鼠源性抗体,可同时识别人和鼠的肿瘤特异抗原。 YlO is directed to a murine antibody specific for human EGFRvIII can simultaneously identify human and murine tumor-specific antigens. 体外研究发现,YIO可以抑制肿瘤DNA合成和细胞增殖,可产生补体或抗体介导的细胞毒作用。 In vitro studies have found, YIO can inhibit tumor cell proliferation and DNA synthesis, can be produced cytotoxicity or complement mediated antibody. 腹腔注射YlO可使稳定表达EGFRvIII的B16 黑色素瘤小鼠动物模型长期生存(η = 20 ; P < 0.001)。 YlO intraperitoneal injection can be stabilized EGFRvIII expressing B16 melanoma mice model of long-term survival of animals (η = 20; P <0.001). YlO在体内作用机制可能依赖于Fc受体。 YlO in vivo mechanism of action may depend on the Fc receptor.

[0046] 单克隆抗体mAb806是用表达EGFRv m的成纤维细胞NR6免疫小鼠来制备的。 [0046] Monoclonal mAb806 was expressing EGFRv m NR6 fibroblasts immunized mice prepared antibody. mAb806显著抑制表达EGFRvIII的U87MG移植荷瘤鼠的肿瘤生长,呈剂量依赖方式;它同时也可抑制EGFR过表达的A431细胞移植荷瘤鼠的肿瘤生长,正常组织中没有EGFRvIII,所含有的肿瘤相关抗正常组织中没有EGFRvIII所含有的肿瘤相关抗原表位,所以此表位可以作为肿瘤疫苗的一个潜在靶标。 mAb806 significantly inhibited the expression of tumor U87MG transplanted mice with tumor EGFRvIII growth in a dose dependent manner; it also inhibit tumor overexpressed EGFR A431 transplanted mice with tumor cell growth, normal tissues do not EGFRvIII, the tumor contained in the relevant no anti normal tissues EGFRvIII tumor associated antigen epitopes contained, so that this epitope can be used as a potential target for tumor vaccine. 针对EGFRvIII抗原表位触发的反应,包括体液免疫与细胞免疫反应。 Epitope against EGFRvIII triggered responses, including humoral and cellular immune responses. eimberger等用单一肽段偶联血蓝蛋白免疫小鼠后,皮下接种肿瘤细胞,结果免疫后小鼠70%未见肿瘤生长,肿瘤生长显著低于对照组(P < 0.05)。 eimberger Once a single conjugated peptide hemocyanin immunized mice, tumor cells were inoculated subcutaneously, 70% results in mice no tumor growth after immunization, tumor growth was significantly lower than the control group (P <0.05). 将免疫鼠血清转移到未免疫鼠体内可以阻止31%小鼠的肿瘤生长P < 0.05),其机制可能与抗体介导的细胞毒作用有关。 The immunized mouse serum was transferred to non-immunized mice can prevent tumor growth in 31% of mice P <0.05), which may be related to the cytotoxicity of antibody-mediated. Ciesielski等将EGFRvIII特异表位(LEEKK-GNYVVTDH)的多个拷贝,用赖氨酸作为桥梁连接起来形成的多重抗原多肽(multiple antigenic peptide)免疫Fisher大鼠,并检测其细胞免疫效应。 Ciesielski like EGFRvIII specific epitope (LEEKK-GNYVVTDH) a plurality of copies, as a bridge to connect together to form a multiple antigenic polypeptide with a lysine (multiple antigenic peptide) Fisher rats immunized, immune effector cells and detected. 多重抗原多肽免疫主要产生特异针对表达EGFRvIII靶抗原的F98胶质瘤细胞的抗肿瘤细胞免疫反应。 Multiple antigenic polypeptide immunogen to produce specific immune response against the main F98 glioma cells expressing EGFRvIII target antigen against tumor cells.

[0047] 在本发明的优选实施例中,首先合成人源EGFRVIII特异表位多肽。 [0047] embodiment, the first synthetic human specific epitope polypeptide EGFRVIII In a preferred embodiment of the present invention. 序列为: CGADSYEMEEDGVRKC。 Sequence: CGADSYEMEEDGVRKC. 该多肽相应抗体已证实对肿瘤治疗有效。 The corresponding polypeptide antibodies have proven effective for tumor therapy. EGFRvIII多肽-PLGA微球构建按实例2方法实施。 EGFRvIII polypeptide -PLGA microspheres embodiment constructed by the method of Example 2. 在通过氨基连接于PLGA/壳聚糖微球(含有BSA+OVA作为免疫载体)。 Connected to PLGA / chitosan microspheres by an amino group (containing BSA + OVA as immunogenic carrier). 按实例1的方法免疫小鼠和测定抗体。 The method according to Example 1 of the immunized mice and assayed for antibody. [0048] 结果显示:6周后EGFRvIII特异表位多肽抗体滴度。 [0048] The results showed that: after 6 weeks EGFRvIII specific epitope polypeptide antibody titer. 微球疫苗获得良好效果, 血清抗体滴度大于32000。 Microsphere vaccine to obtain good results, serum antibody titers greater than 32,000.

[0049] 根据本发明的一个优选实施例,提供一种表皮生长因子受体HER2/neu构建的免疫微球。 [0049] According to a preferred embodiment of the present invention, there is provided an epidermal growth factor receptor HER2 / neu construct immune microspheres. HER2/neu是表皮生长因子受体家族的第二位成员。 HER2 / neu is the second member of the epidermal growth factor receptor family. 表皮生长因子受体(EGFR) 家族,也称为HER或ErbB2家族,在细胞信号转导中发挥重要作用,是细胞生长、分化及存活的重要调节者。 Epidermal growth factor receptor (EGFR) family, also known as ErbB2 or HER family, plays an important role in cellular signal transduction, cell growth, differentiation and survival important regulator. 此家族包括EGFR (HERI或ErbBi)、HER2/neu (neu或ErbB2)、 HER3(ErbB3)及HER4(tyro2或Erb_B4),人HER2/neu基因定位于17号染色体短臂,表达产物为分子量185kDa的单链跨膜糖蛋白,含有1255个氨基酸残基,细胞内段(ICD) 具有酪氨酸激酶活性。 This family consists of EGFR (HERI or ErbBi), HER2 / neu (neu or ErbB2), HER3 (ErbB3) and HER4 (tyro2 or Erb_B4), human HER2 / neu gene chromosomal location at 17 short arm, the expression product molecular weight 185kDa of single-chain transmembrane glycoprotein containing 1255 amino acid residues, intracellular segment (ICD) having a tyrosine kinase activity. HER2/neu通过多种细胞内分子参与信号传导,其主要下游物质包括丝裂原活化已知有9种配体能直接与EGFR、HER3或HER4结合,在组织中EGFR家族成员很少单独表达.而是形成不同组台,构成同源或异源二聚体,放大信号转导而发挥生物学作用由HER2/neu组成的受体复合物比其他受体联合更有效。 HER2 / neu by a variety of intracellular molecules involved in signal transduction, which comprises a main downstream mitogen-activated species there are nine known ligand binds directly with EGFR, of HER3 or HER4, EGFR family members in the tissue in very little expression alone. but form different sets of stations constituting a homologous or heterologous dimers, amplified signal transduction and biological effects play a more effective by the HER2 / neu receptor complex consisting of the joint than the other receptors. 因为①HER2受体复合物具有较高的配体结合能力,且内化率低能在浆膜上停留更长时间,延长受体信号传导的持续时间;②HER2是一种非常活跃的酪氨酸激酶,其自身突变及过度表达也能产生结构上活化。 Because ①HER2 receptor complex has a high ligand binding capacity, and the rate is low can stay longer in the plasma membrane, extending the duration of receptor signaling; ②HER2 is a highly tyrosine kinase, overexpression and mutation itself can be generated on the activation structure. 通常情况下,HER2/neu只在胎儿时期表达,到成年以后,用免疫组化染色只能在极少组织内发现其低水平表达于细胞表面。 Typically, HER2 / neu is expressed only in the prenatal period, after the adult, by immunohistochemical staining can only be found in very few tissues which expressed low levels of the cell surface. 在人类癌症,HER2/neu的分子改变通常是正常基因产物的过度表达。 In human cancers, HER2 / neu is overexpressed molecular changes usually normal gene product. 多种人类癌症存在HER2/neu过度表达,如乳腺癌(25〜30)、卵巢癌(25〜32)、肺腺癌(30〜35)、原发性肾细胞癌(30〜40)等由于HER2/neu蛋白定位于浆膜,即使中等水平HER2过度表达也能产生受体结构上活化。 The presence of a variety of human cancers HER2 / neu overexpression, such as breast cancer (25~30), ovarian cancer (25~32), lung adenocarcinoma (30~35), primary renal cell carcinoma (30 to 40) or the like due to the HER2 / neu protein located in the plasma membrane, even moderate levels of overexpression of HER2 activation can be generated on the receptor structure. 免疫组化染色发现乳腺癌细胞的HER2蛋白水平比邻近正常乳腺上皮高10〜100倍。 Immunohistochemical staining HER2 protein levels found in breast cancer cells 10~100 times higher than adjacent normal breast epithelium. 人类肿瘤常见HER2/neu扩增及过度表达,表明HER2/neu在肿瘤发生中起重要作用。 Common human tumor HER2 / neu amplification and overexpression, suggesting that HER2 / neu plays an important role in tumorigenesis. HER2/ neu的过度表达通过启动多种转移相关机制而增加转移能力.包括细胞迁移率、体外侵袭力、IV型腔原酶活性、实验性肺转移等。 HER2 / neu is overexpressed by activating more transfer mechanisms to increase transfer capability, including the rate of cell migration, invasion in vitro, IV cavity reductase activity, experimental lung metastasis. HER2/rieu过度表达也影响某些粘附分子如上皮细胞钙粘蛋白(E-cadherin)等合成.从而促进转移。 HER2 / rieu overexpression also affect the synthesis of certain adhesion molecules such as epithelial cadherin (E-cadherin) and other cells to facilitate the transfer. HER2/neu过度表达乳腺癌细胞对雌激素治疗不敏感。 HER2 / neu-overexpressing breast cancer cells are not sensitive to estrogen therapy. HER2/neu过度表达肿瘤细胞对某些化疗药物治疗也不敏感。 HER2 / neu over-expressing tumor cells are not sensitive to certain chemotherapy drugs. Tsai 等人在20种非小细胞肺癌细胞系中发现原发化疗耐药与HER2/neu表达显著相关。 Tsai et al found that 20 kinds of non-small cell lung cancer cell lines, primary chemoresistance significantly associated with HER2 / neu expression. HER2 过度表达诱导乳腺癌细胞对紫杉醇、紫杉萜耐药。 HER2 over-expressing breast cancer cells induced by paclitaxel, docetaxel resistance. HER2/neu蛋白是癌症主动免疫治疗的理想靶点。 HER2 / neu protein is an ideal target for active immunotherapy of cancer. 因为HER2/neu蛋白与细胞恶性转化有关,是癌症病因学中一种生物学相关蛋白;某些肿瘤病人存在HER2/neu特异性抗体,表明HER2/neu疫苗能诱导免疫反应; 已证明被动免疫疗法如HER2/neu抗体抗肿瘤作用,预测主动免疫可能也是有效的。 Because HER2 / neu protein and cell malignant transformation is a biological etiology of cancer-associated protein; there are some cancer patients HER2 neu specific antibody /, indicating that HER2 / neu vaccine to induce an immune response; passive immunotherapy has been demonstrated the HER2 / neu antibody anti-tumor effect, active immunization prediction may also be effective.

[0050] 在本发明的一个优选实施例中,首先合成人源HER2特异表位多肽CQMWAPQ WGPDC O针对该多肽的特异抗体已被证实对肿瘤治疗有效。 [0050] embodiment, the first synthetic human HER2 specific epitope polypeptide CQMWAPQ WGPDC O has been proven effective in the treatment of tumor specific antibodies against the polypeptide of the present invention in a preferred embodiment. 免疫微球构建材料为琼脂糖。 Immune build material agarose microspheres. 微球含有BSA+OVA作为免疫载体,多肽抗原用戊二醛通过氨基连接于琼脂糖/壳聚糖微球。 BSA + OVA containing microspheres as immunogenic carrier, with glutaraldehyde polypeptide antigen linked to agarose / chitosan microspheres by an amino group.

[0051] 结果显示:6周后Her2特异表位多肽构建的免疫微球免疫小鼠血清抗体滴度大于32000。 [0051] The results show: Her2 polypeptide construct specific epitope 6 weeks after vaccination serum antibody titers microsphere immunized mice were greater than 32,000. 比常规BSA_Her2方法高5倍以上。 Higher than conventional methods BSA_Her2 more than 5 times.

附图说明 BRIEF DESCRIPTION

[0052] 图1是免疫微球模拟图,其中1表示免疫载体蛋白,2表示疫苗抗原。 [0052] FIG. 1 is a mimetic diagram immune microspheres, wherein 1 represents immunogenic carrier protein, 2 denotes a vaccine antigen. [0053] 图2是表皮生长因子构建的微球免疫小鼠血清中EGF抗体测定结果图,其中横坐标表示血清稀释比例,纵坐标表示OD450值,每组柱状图中的A表示实验组,B表示对照组,C表示空白。 [0053] FIG. 2 is a measurement result of FIG EGF antibodies in serum of immunized mice microspheres of Epidermal Growth Factor in construction, where the abscissa represents the serum dilution ratio, the ordinate represents the OD450 values, each set of columns indicates the experimental groups A, B represents the control group, C represents a blank.

[0054] 图3是表皮生长因子(EGF)构建的微球的West-blot检测结果图,其中3表示BSA, 4 表示EGF。 [0054] FIG. 3 is a microsphere of epidermal growth factor (EGF) constructed FIG West-blot detection result, wherein 3 represents BSA, 4 indicates EGF.

具体实施方式 Detailed ways

[0055] 下面结合附图和具体实施例进一步说明本发明的技术方案,但本发明不仅局限于实施例。 [0055] The following further illustrate the technical solutions of the present invention in conjunction with the accompanying drawings and the specific embodiments, but the present invention is not limited to the embodiment.

[0056] 实施例1包裹牛血清白蛋白的硅藻酸钙/壳聚糖微球制备 [0056] Example 1 Calcium diatom wrapped bovine serum albumin / chitosan microspheres prepared

[0057] 一、包裹牛血清白蛋白(BSA)的硅藻酸钙微球制备 [0057] I. Preparation of bovine serum wrapped albumin (BSA) microspheres calcium diatom

[0058] 海藻酸钠(sodium alginate) 2g溶于90ml双蒸水中,加牛血清白蛋白(BSA)至终浓lmg/ml。 [0058] Sodium alginate (sodium alginate) 2g dissolved in 90ml double distilled water, add bovine serum albumin (BSA) to a final concentration of lmg / ml. 0.5M NaOH调PH至7.3,加水定容至IOOml配置成2% (m/v)溶液。 0.5M NaOH adjust PH to 7.3, add water to IOOml configured to 2% (m / v) solution. 氯化钙(CaC12)配制成10% (m/v)溶液。 Calcium chloride (CaCl2) formulated as a 10% (m / v) solution. 2%海藻酸钠溶液4ml气泵喷笔雾化喷入IOOml氯化钙溶液中,500转/分磁力搅拌成球。 4ml of 2% sodium alginate solution into atomizing pump Airbrush IOOml calcium chloride solution, 500 rev / min with magnetic stirring into a ball. 磁力搅拌平衡lh,离心收集微球,双蒸水搅拌平衡lh,离心洗涤3次。 Balanced magnetic stirring lh, microspheres were collected by centrifugation, centrifugation was washed with double-distilled water was stirred for lh balance 3 times.

[0059] 显微镜检视大小和微球均一性。 [0059] Now microscope and microsphere size uniformity. 根据红细胞大小对照判断微球直径约为5-8微米,显微镜观察分散性良好。 The erythrocyte size control of microspheres is determined diameter of about 5-8 micrometers, microscopic observation of good dispersibility.

[0060] 相同方法制备不含BSA的海藻酸钙微球用于对照试验。 Preparation of BSA-free [0060] the same manner as alginate microspheres for controlled trials. 微球直径大小与上述相似。 Diameter microspheres similar to the above. 显微镜观察分散性良好 Microscopic observation good dispersibility

[0061] 二、微球中BSA含量测定 [0061] II. Determination of BSA microspheres

[0062] Iml微球悬浮于5ml、0.1M、pH7.6的磷酸缓冲液中。 [0062] Iml microspheres are suspended in 5ml, phosphate buffer 0.1M, pH7.6 in. 二小时后海藻酸钙微球解体溶介。 Calcium alginate beads disintegrated after two hours for dissolving. BSA释放于溶液中,测定BSA的紫外光吸收OD2SO。 BSA in the release solution, the ultraviolet absorption was measured BSA OD2SO. 浓度lmg/ml的OD28tl 值为0.56。 A concentration of lmg / ml in 0.56 OD28tl. 按此推算,微球中BSA浓度约为0.75mg/ml。 This calculation, the microspheres BSA concentration of about 0.75mg / ml.

[0063] 三、包复壳聚糖的海藻酸钙微囊制备 [0063] Third, calcium alginate microcapsules prepared chitosan cladding

[0064] 该步骤的目的是在包裹蛋白的外表面引入可以偶联蛋白的氨基,同时用壳聚糖包复微球表面克服蛋白从微球中漏出 [0064] The purpose of this step is to introduce an amino group can be conjugated to proteins on the outer surface of the envelope proteins, while overcoming protein clad with chitosan microsphere leaking from the microspheres

[0065] 壳聚糖(chitosan)MW : 18000〜20000取0.5g溶于醋酸溶液,定容至100ml,终浓度为0.5% (m/v)。 [0065] Chitosan (chitosan) MW: 18000~20000 dissolved in 0.5g of acetic acid solution volume to 100ml, final concentration of 0.5% (m / v).

[0066] 藻酸钙微球与0.5%壳聚糖溶液混合,室温磁力搅拌4小时。 [0066] Calcium alginate beads was mixed with 0.5% chitosan solution, magnetically stirred for 4 hours at room temperature. 离心收集微囊,振荡器打散缓慢加入生理盐水至悬液,离心收集微囊,洗涤重复5次。 Microcapsules were collected by centrifugation, physiological saline was added slowly to break the oscillator suspension, the microcapsules were collected by centrifugation, washing was repeated 5 times.

[0067] 包复壳聚糖的微球分散性较海藻酸钙微球略差但满足实验要求。 [0067] Microspheres Chitosan clad dispersibility than alginate beads but slightly inferior to meet the test requirements.

[0068] 四、聚糖氨基定量 [0068] Fourth, the glycan quantitative amino

[0069] 1.三硝基苯磺酸钠(TNBS)可与壳聚糖中游离氨基产生橙红色反应,加入终止液后335nm比色。 [0069] 1. trinitrobenzene sulfonate (the TNBS) can produce an orange-red free amino group is reacted with the chitosan, 335nm after adding stop solution color. 按消光系数(E335 = 14000)计算氨基浓度 Press extinction coefficient (E335 = 14000) amino group concentration calculated

[0070] 2.TNBS (5% m/v)稀释到0.01% (m/v)工作液。 [0070] 2.TNBS (5% m / v) was diluted to 0.01% (m / v) working solution. 反应缓冲液为PH8.50.1M碳酸 The reaction buffer was PH8.50.1M carbonate

缓冲液。 Buffer.

[0071] 3.不含有BSA的微球用于氨基测定,因蛋白氨基同时显色。 [0071] 3. BSA microspheres containing no amino group for measuring, at the same time due to protein amino color. 微球体积定量:微球悬生理盐水,用带刻度的离心管中1000转/分离心。 Quantitative volume of the microspheres: Microspheres suspended physiological saline, with a graduated centrifuge tube 1000 rpm / centrifuged. 观察离心后的微球体积(以下称之谓压积),然后根据需要稀释到需要浓度。 Observation volume of the microspheres (hereinafter known that the hematocrit), and then diluted to the desired concentration if necessary after centrifugation. 以下微球的百分浓度均为压积百分浓度 The following percent concentrations of microspheres are hematocrit percentage concentration

[0072] 4、10%微球样品取样2ml,加入0.01 % (m/v) TNBS 0.5ml,于37°C水浴2h,再加入0.5ml 10% SDS和0.25ml IM盐酸终止。 [0072] 4,10% microsphere sample sampling 2ml, was added 0.01% (m / v) TNBS 0.5ml, at 37 ° C water bath for 2h, then added 0.5ml 10% SDS and 0.25ml IM hydrochloric acid terminated. 5分钟后于335nm比色。 5 minutes after the colorimetric 335nm. 测得OD335 = 0.75。 Measured OD335 = 0.75.

[0073] 5、氨基估算(0.75+ 14000 X 6.023 X IO23) + 微球数/ 升 [0073] 5, amino estimate (0.75+ 14000 X 6.023 X IO23) + Micro / liter ball

[0074] 海藻酸钙微球接近红细胞大小,估算约为2000万/μΐ,即按2Χ1013/升计算。 [0074] The alginate microspheres close to the red blood cell size, estimates of about 20 million / μΐ, i.e. press 2Χ1013 / liter calculated. 每个微球估算大约有150000个氨基。 Each microsphere amino estimated about 150,000. 以下微球氨基测算均以此方法。 The following amino calculation microspheres are in this way.

[0075] 实例2、包裹卵白蛋白(OVA)的聚乳酸-乙醇酸(PLGA)/壳聚糖微球制备 [0075] Example 2, wrapped ovalbumin (OVA) polylactic acid - glycolic acid (PLGA) / chitosan microspheres prepared

[0076] 本实施例中所采用的微球制备操作可参考文献:(1)载药微球的制备、表征与体外缓释研究;清华大学研究生论文(2008)王逸蔚,崔福斋。 [0076] The operation of the present microspheres prepared in Example embodiments may be employed in References: (1) Preparation of drug-loaded microspheres, characterization and in vitro release studies; Tsinghua University graduate thesis (2008) Yi Wang Wei, Cui Fuzhai. (2)聚乳酸微球的制备; 吉林大学学报(理学版)V ol.43N ο.6Νον 2005 : 842-846。 Preparation of polylactic acid microspheres (2); Jilin University (Science Edition) V ol.43N ο.6Νον 2005: 842-846.

[0077] 一、聚乳酸-乙醇酸共聚物购于SIGMA公司(成份比为50 : 50),IOOmg溶于5mL有机溶剂二氯甲烷中。 [0077] a polylactic acid - glycolic acid copolymer available from SIGMA Company (component ratio 50: 50), IOOmg 5mL dissolved in an organic solvent such as dichloromethane. 秤取5mgOVA,干粉用细菌研磨器将OVA颗粒研细。 Weighed 5mgOVA, bacterial dry grinding will finely particulate OVA. 悬浮于含有PLGA的有机溶剂。 Suspended in an organic solvent containing the PLGA. 并缓慢倒入50mL含聚乙烯醇(PVA)的水相中,冰浴下以300W功率超声乳化30s。 And slowly poured into 50mL containing polyvinyl alcohol (PVA) aqueous phase in an ice bath phacoemulsification power 300W 30s. 随后将复乳倒入150mL双蒸水中常压磁力搅拌5h以挥发有机溶剂,15000r/min离心IOmin收集,去离子水洗涤3次,冷冻干燥48h去除微球中水分,于-20°C保存。 The double emulsion is then poured into 150mL atmospheric magnetic stirring 5h double distilled water to volatilize the organic solvent, 15000r / min IOmin collected by centrifugation, washed with deionized water three times and freeze-dried microspheres 48h remove water and stored at -20 ° C.

[0078] 显微镜下观察微球粒径,根据视觉粒径及显微放大倍数判断,重复制备的微球平均粒径均为2.0〜3.0 μ m。 [0078] The microsphere size was observed under a microscope, magnification is determined based on the visual and microscopic particle size, average particle diameter of the microspheres prepared are repeated 2.0~3.0 μ m. 分散性良好 Good dispersion

[0079] 二、微球包裹OVA含量测定 [0079] II. Determination of microsphere inclusions OVA

[0080] Iml用于测定的微球悬于3ml 二氯甲烷,轻轻震摇使微球溶解,加入3ml0.1M、 pH7.2的磷酸缓冲液溶介OVA。 [0080] Iml assay for microspheres suspended in 3ml of dichloromethane, and shaken gently to dissolve the microspheres, was added 3ml0.1M, pH7.2 phosphate buffer for dissolving OVA. 离心取上层水相测定OD280。 The upper phase assay OD280 centrifuged water. lmg/ml为E2800.74。 lmg / ml to E2800.74. 结果显示,PLGA微球中OVA浓度约为0.9mg/ml The results show, PLGA microspheres OVA concentration of about 0.9mg / ml

[0081] 三、PLGA微球包复壳聚糖。 [0081] III, PLGA microspheres chitosan cladding. 操作条件如实例1。 Operating conditions as described in Example 1. TNBS比色法测定氨基,估算大约每亇微球有30000个氨基 TNBS colorimetry amino estimated approximately every 30,000 Ma microspheres amino

[0082] 四、包裹BSA的PLGA微球操作条件与上相同,仅用BSA取代OVA蛋白。 [0082] Fourth, the package BSA PLGA microspheres on the same operating conditions, with only BSA substituted OVA protein.

[0083] 五、包裹BSA+OVA的PLGA微球操作条件与上相同,用BSA+OVA蛋白代替单 [0083] Fifth, the wrapped BSA + PLGA microspheres operating conditions of OVA on the same, instead of a single protein BSA + OVA

一蛋白。 A proteins.

[0084] 实例3包裹BSA的琼脂糖微球制备 [0084] Example 3 Preparation of BSA-agarose beads wrapped

[0085] 一、制备方法:低凝固温度琼脂糖购于SIGMA公司。 [0085] I. Preparation: low freezing temperature agarose available from SIGMA Company. 琼脂糖用蒸馏水加热熔化,浓度为4%。 Heating and melting the agarose with distilled water, concentration of 4%. 执置于45C度水浴。 Executive placed in 45C degree water bath. BSA溶解于0.05MPH7.2磷酸缓冲液中,浓度为0.2%。 0.05MPH7.2 BSA was dissolved in phosphate buffer at a concentration of 0.2%. 等体积蛋白溶液与琼脂糖混合使最终浓度:琼脂糖为2%,蛋白浓度为与0.5%。 An equal volume of protein solution was mixed with agarose final concentration: 2% agarose, and protein concentration of 0.5%.

[0086] 微球制备用膜乳法制备,其具体操作可参考文献:快速膜乳化法制备粒径均一的PLGA微球和微囊。 [0086] Preparation of microspheres prepared by film emulsion method, the specific operation may Reference: Membrane Emulsification Method Preparation of Uniform Size PLGA microspheres and microcapsules. 田瑞,王连艳等。 Tian Rui, Wang Lianyan and so on. 过程工程学报Vol.9No.4。2009年8月。 Process Engineering Vol.9No.4. 2009 in August 754-761。 754-761.

[0087] 膜乳化法制备微球是将溶化的琼脂糖在高压不通过陶瓷膜微孔(孔径0.1-5微米)分散进入油相形成微球。 [0087] Preparation of membrane emulsification microspheres are formed with the melting agarose beads in a high pressure without passing through the porous ceramic membrane (pore diameter 0.1-5 microns) was dispersed into the oil. 油相成份为含有2% SPAN80的食用豆油。 An oil phase component containing 2% SPAN80 of edible soybean oil. 陶瓷膜孔径为0.5微米,乳化时油温保持45度。 Ceramic membrane pore size of 0.5 microns, emulsified oil temperature remains at 45 degrees. 磁力搅拌下获得1-3微米的含有蛋白的琼脂糖微球。 Under magnetic stirring to obtain a protein containing 1-3 microns agarose beads. 撤去加热后使冷却到室温使琼脂糖微球凝固。 After the heating was removed and cooled to room temperature so that solidification of agarose beads. 离心收集微球。 Microspheres collected by centrifugation. 微球用25-75%逐步增加酒精浓度在低温下(0-5度)洗涤除去油相成份并固定蛋白。 Microspheres with a gradual increase in ethanol concentration 25-75% at low temperature (0-5 degrees) is washed to remove the oil phase component and immobilized protein.

[0088] 二、微球镜检结果:根据视觉粒径及显微放大倍数判断,制备的微球平均粒径均为2.0-3.0 μ m。 [0088] Second, the microspheres Microscopic examination: The particle size of the visual and microscopic magnification is determined, the average particle size of microspheres prepared are 2.0-3.0 μ m. 分散性良好。 Good dispersion.

[0089] 三、蛋白含量测定 [0089] Third, the determination of the protein content

[0090] IOOmg乙醇干燥的微球悬浮于5.0ml磷酸缓冲液(0.05M pH7.2)中。 [0090] IOOmg ethanol dried microspheres suspended in 5.0ml of phosphate buffer (0.05M pH7.2) in. 冰箱中放置48小时使蛋白充分释放于溶液中。 For 48 hours to fully release the protein in the solution in a refrigerator. OD2SO测定卵白蛋白含量。 Determination of ovalbumin OD2SO content. 结果显示每IOOmg干微球含卵白蛋白2.5mg The results showed that each IOOmg dry microspheres containing ovalbumin 2.5mg

[0091] 四、包的复壳聚糖的结果 [0091] Fourth, the results of the complex chitosan packet

[0092] 方法与实例3相同,用不含蛋白的微球做包复壳聚糖氨基测定(蛋白干扰氨基测定)。 [0092] the same manner as in Example 3, amino assay do wrap chitosan (amino interference protein assay) using protein-free microspheres. 估算每亇微球(按平均直经2.5微米计)含有壳聚糖氨基大于30000个 Each estimate Ma microspheres (average straight through 2.5 microns) containing chitosan amino greater than 30,000

[0093] 五、包裹OVAA的琼脂微球操作条件与上相同,仅用OVA取代BSA蛋白。 [0093] V. agar microspheres OVAA the operating conditions in the same package, with only OVA substituted BSA protein.

[0094] 六、包裹BSA+OVA的琼脂微球操作条件与上相同,用BSA+OVA蛋白代替单一蛋白。 [0094] VI wrapped agar BSA + OVA microspheres operating conditions of the same, instead of a single protein with BSA + OVA protein.

[0095] 实例4微球与抗原蛋白的连接一戊二醛方法连接 [0095] Example 4 antigen protein microspheres glutaraldehyde method for connecting a connector

[0096] 一、以EGF为例说明微球与抗原的偶联 [0096] First, an example to EGF antigen coupled microspheres

[0097] 二、取洗涤过的海藻酸钙/壳聚糖微球离心压积0.5ml重悬于2.5ml的碳酸缓冲液(0.1M、pH8.5)。 [0097] Second, to take the washed calcium alginate / chitosan microspheres hematocrit by centrifugation and resuspended in 0.5ml 2.5ml of the carbonate buffer (0.1M, pH8.5). 加入2.5ml 0.5%戊二醛,室温混合2h活化后离心收集洗涤3次。 After addition of 2.5ml 0.5% glutaraldehyde, mixed at room temperature 2h collected by centrifugation and washed three times with activation.

[0098] 三、微球重新悬于3ml的碳酸缓冲液中。 [0098] Third, the microspheres are resuspended in 3ml of carbonate buffer. 加入500微克小鼠来源mEGF (sigma 公司)。 Add 500 [mu] g origin mEGF (sigma Corporation) mice. 置于混合器上冰箱中混合反应4小时后,加入乙醇胺100微升封闭余下戊二醛基团。 After the reaction mixture was placed on the mixer in a refrigerator for 4 hours, 100 microliters of ethanolamine blocking groups remaining glutaraldehyde. 然后用生理盐水洗涤三次。 Then washed three times with physiological saline.

[0099] 四、微球偶联EGF结果: [0099] Fourth, EGF microspheres conjugated Results:

[0100] 抗小鼠EGF抗体、辣根过氧化物酶标记的亲和素(Avidin) TMB显色底物工作液购于武汉新启迪生物科技有限公司。 [0100] Mouse anti-EGF antibody, horseradish peroxidase labeled avidin (Avidin) TMB chromogenic substrate working solution purchased from Wuhan inspiration biotechnology company. EGF-海藻酸钙微球悬浮于试管中,按操作说明书相继加入Biotin-EGF抗体、HRP_avidin、TMB。 EGF- alginate microspheres are suspended in a test tube, according to operating instructions sequentially added Biotin-EGF antibody, HRP_avidin, TMB. 用肉眼及显微镜可观察到微球显兰色。 It was observed with the naked eye and the microscope significant blue microspheres. [0101 ] 实例5EGF微球疫苗动物试验结果 [0101] Examples of microsphere vaccine 5EGF animal test results

[0102] 一.动物免疫 [0102] a. Animal immunization

[0103] 1.20只昆明种小鼠随机分为3组:实验组8只,对照组8只,空白组4只。 [0103] 1.20 Kunming mice were randomly divided into three groups: the experimental group 8, group 8, group four blank.

[0104] 2.免疫微囊以生理盐水重悬。 [0104] 2. A microcapsule immune resuspended with saline. 实验组注射含BSA的微囊,2% (ν/ν)微囊0.1ml 尾经脉注射;10% (V/V)0.2ml背部皮下多点免疫注射。 The experimental group injected with BSA microcapsules, 2% (ν / ν) microcapsules injected 0.1ml tail meridian; 10% (V / V) 0.2ml subcutaneously multipoint immunization. 对照组注射不含BSA的微囊, 方法同上。 BSA-free control group was injected microcapsules as above. 空白组注射生理盐水,方法同上。 Blank group was injected with saline as above.

[0105] 3.初免后14d再次免疫一次,10日后小鼠处死,眼球采血,分离血清。 [0105] 3. First Free 14d after immunization once again, the mice were sacrificed 10 days, eye blood serum was separated.

[0106] 二.ELISA 检测抗体 [0106] bis detection antibody .ELISA

[0107] l.mEGF和BSA分别以0.05M PH9.6碳酸缓冲液配制20ug/ml包被液,0.1ml每 [0107] l.mEGF respectively BSA and 0.05M PH9.6 carbonate buffer solution 20ug / ml coating buffer, 0.1ml per

孔过夜包被酶标板。 ELISA plates were coated overnight hole.

[0108] 2.PBST洗涤3次,每孔以0.3ml无蛋白封闭液室温封闭2h。 [0108] 2.PBST washed 3 times with 0.3ml per well blocking solution at room temperature protein free blocked 2h.

[0109] 3.洗涤3次分别加入梯度稀释的小鼠血清各0.1ml,37°C孵育2h。 [0109] 3. Wash 3 times serial dilutions of mouse serum were added to each 0.1ml, 37 ° C incubation 2h.

[0110] 4.PBST洗涤5次加入1 : 2000兔抗鼠-HRP每孔0.1ml,室温孵育lh。 [0110] 4.PBST washed 5 times with 1: 2000 rabbit anti-mouse -HRP each well 0.1ml, incubated at room temperature lh. 洗涤5 次后分别加入O.lmlTMB显色,显色15min各加入50ul IM硫酸 After 5 washes were added O.lmlTMB color, color 15min sulfuric acid was added to each 50ul IM

[0111] 三、动物试验结果[0112] EGF抗体测定结果见图2。 [0111] Third, the results of animal studies [0112] EGF antibody assay results shown in Figure 2. EGF抗体平均滴度为8000。 EGF antibody titers average of 8,000. [0113] 四1West—blot检测[0114] 1. [0113] IV 1West-blot detection of [0114] 1. mEGF和BSA分别以20ug上样做SDS—PAGE电泳。 BSA and respectively on mEGF doing 20ug SDS-PAGE electrophoresis. [0115] 2.100mA恒流转膜lh。 [0115] 2.100mA constant flow membrane lh. [0116] 3. [0116] 3. 无蛋白封闭液封闭2h。 Protein-blocking solution 2h. [0117] 4.1:500小鼠血清37℃结合2h。 [0117] 4.1: 37 [deg.] C 500 mouse serum binding 2h. [0118] 5. [0118] 5. PBST洗涤5次加入l:2000兔抗鼠一HRP,室温孵育lh。 Washed with PBST 5 times was added l: 2000 a rabbit anti-mouse HRP, incubated at room temperature lh. [0119] 6. [0119] 6. 洗涤5次,加入DAB显色[0120] 7. Washed 5 times, DAB color was added [0120] 7. 结果如图3,实验组小鼠产生抗BSA和mEGF抗体。 The results in FIG. 3, the experimental group of mice and the anti-BSA antibodies mEGF. 在BSA及EGF电泳位置显色。 EGF and BSA in color electrophoretic position. [0121] 实施例6血管生长因子(VEGF)构建的微球[0122] 一1鼠源血管内皮细胞生长因子(mVEGF)购于sigma公司。 [0121] Example 6 Construction of vascular endothelial growth factor (VEGF) microspheres Embodiment [0122] 1 a murine vascular endothelial growth factor (mVEGF,) purchased from sigma company. PLGA/壳聚糖微球制备见实例2。 PLGA / chitosan microspheres prepared as described in Example 2. mVEGF与微球的偶联用戊二醛方法连接。 mVEGF microspheres with glutaraldehyde coupling method is connected. 见实施例4。 See Example 4. [Ol 23] 二1偶联结果鉴定[0124] 用于鉴定异硫氢酸荧光素标记兔抗小鼠VEGF多克隆抗体,购于上海晶天生物科技有限公司。 [Ol 23] The results identified two coupling 1 [0124] for identifying acid fluorescein isothiocyanate labeled rabbit anti-mouse polyclonal antibody VEGF, purchased from Shanghai days crystal biotechnology company. [0125] 按说明书操作用荧光标记抗体检测微球表面的mVEGF。 [0125] mVEGF antibody labeled by manual operation of the surface of the microspheres with fluorescence detection. 用荧光显微镜观察结果。 With a fluorescence microscope. 在荧光显微镜视野下可见微球发明亮荧光。 In the field fluorescence microscopic beads made bright fluorescence. [0126] 实验小鼠(昆明种,来自上海实验动物中心)分四组,每组七只小鼠[Ol 27] l1lo%mVEGF一微球(PLGA微球包含有OVA)静脉注射免疫[0128] 2110%mVEGF一微球(PLGA空白微球)静脉注射免疫[0129] 31lo ug mVEGF用Friend佐剂常规皮下注射免疫[Ol 30] 41没有任何处理的小鼠血清为阴性血清对照[0131] 低速离心压积为o. [0126] mice (Kunming, from Shanghai Experimental Animal Center) divided into four groups, each group of seven mice [Ol 27] l1lo% mVEGF a microsphere (PLGA microspheres with OVA) intravenous immunoglobulin [0128] 2110% mVEGF a mouse serum microspheres (PLGA microspheres blank) intravenous immunoglobulin [0129] 31lo ug mVEGF subcutaneous immunization with a conventional adjuvant Friend [Ol 30] 41 without any treatment as negative control sera [0131] low speed centrifugation hematocrit for the o. 5ml免疫微球悬浮于5ml PBS中。 5ml immune microspheres are suspended in 5ml of PBS. 注射量为100微升第一次免疫后周后加强免疫,以后每周加强一次。 The injection volume was 100 microliters for the first time after booster immunization Later Zhou Dynasty, after strengthening once a week. 六周后摘眼球取血。 After six weeks eyeball blood. 常规ELISA方法测定血清中抗体滴度。 A conventional ELISA method for measuring serum antibody titer. 结果见表l[Ol 32] 表1VEGF微球免疫小鼠血清抗体测定(OD450±SD)[0133] The results are shown in Table l [Ol 32] Table 1VEGF serum antibody microsphere immunized mice (OD450 ± SD) [0133]

Figure CN102008719AD00121

[0134] 以小鼠VEGF为抗原免疫动物,免疫6周后获得l:64000的滴度。 [0134] In mouse VEGF as antigen to immunize an animal, 6 weeks after immunization obtain l: 64000 titer. 满足进一步研究抗肿瘤研究的技术基础。 Meet the technical basis for further study of anti-cancer research. [0135] 实施例7小鼠IgE C s 3肽构建的微球[0136] 一1包裹牛血清白蛋白(BSA)的PLGA/壳聚糖微球制备见实施例2。 [0135] Example 7 Mouse IgE C s 3 peptide constructed microspheres embodiment [0136] 1 a wrapped bovine serum albumin (BSA) in PLGA / chitosan microspheres prepared as in Example 2. [0137] 二1 小鼠IgE C s 3结构域抗原肽按以下文献化学合成, 其合成方法参考文献(Synthetic IgE peptide vaccine for immunotherapy of allergy Vaccine21 (2003) 1580-1590Chang Yi Wang, Alan M.Walfield, Xinde Fang, Bruce Hammerberg, JohnYe) [0137] bis mouse IgE C s 3 1 domain antigen peptide chemically synthesized according to the following literature references synthesis method (Synthetic IgE peptide vaccine for immunotherapy of allergy Vaccine21 (2003) 1580-1590Chang Yi Wang, Alan M.Walfield, Xinde Fang, Bruce Hammerberg, JohnYe)

0138]小鼠 IgE C ε 3 结构域序列为:GYGYQCIVDHPDFPKPIVRSITKTPGQR。 0138] IgE C ε domain sequences of 3 mice: GYGYQCIVDHPDFPKPIVRSITKTPGQR.

0139] 三、C ε 3肽与BSA交朕委托公司用常规戊二醛方法制备。 0139] III, C ε 3 peptide I cross-BSA commissioned glutaraldehyde prepared by conventional methods. 据公司提供数据每个BSA连接多肽在3.5分子、 According to data provided by connecting each polypeptide in 3.5 BSA molecule,

0140] 0140]

0141] 0141]

0142] 0142]

0143] 0143]

0144] 0144]

0145] 0145]

0146] 0146]

0147] 0147]

0148] 0148]

0149] 0149]

0150] 0150]

0151] 0151]

0152] 0152]

0153] 0153]

组别 Group

阴性血清 Negative serum

C ε3-BSA C ε 3-微球Ce3 C ε3-BSA C ε 3- microspheres Ce3

实施例8小鼠IgE C ε 3构建的微球的动物试验结果 Animal test results Example 3 Mouse IgE C ε 8 embodiment constructed microspheres

一、动物试验分组(每组七只)昆明种小鼠购自上海实验动物中心 An animal test groups (seven per group) were purchased from Shanghai Kunming mice Experimental Animal Center

1、C ε 3肽-BSA,Friend佐剂皮下免疫 1, C ε 3 peptide -BSA, Friend adjuvant subcutaneously immunized

2、C ε 3肽-微球尾静脉注射免疫、 2, C ε 3 peptide - intravenous injection of microsphere immunized,

3、C ε 3肽Friend佐剂皮下免疫 3, C ε 3 peptide adjuvant subcutaneously immunized Friend

4、未经任何处理的小鼠血清 4, without any treatment of mouse serum

二、免疫0.5毫升免疫微球悬浮于5mlPBS中。 Second, the immunization immunization 0.5 ml of microspheres suspended in 5mlPBS. 尾静脉注射量为100微升第一次免疫后二周后加强免疫,以后每周加强一次。 Tail vein injection volume of 100 microliters after the first booster immunization after two weeks, once a week after strengthening. 六周后摘眼球取血。 After six weeks eyeball blood. C ε 3肽-BSA 20微克与Friend佐剂混合皮下注射。 C ε 3 -BSA 20 [mu] g peptide mixture Friend adjuvant and injected subcutaneously. 程序与上相同 Program on the same

C ε 3肽20微克与Friend佐剂混合,皮下注射免疫。 C ε 3 peptide was mixed with 20 g Friend adjuvant, subcutaneous immunization. 程序与上相同 Program on the same

三、抗体滴度测定:ELISA常规方法 Third, the antibody titer assay: ELISA conventional methods

常规ELISA方法测定血清中抗体滴度体测定结果如表2 表2IgE微球免疫小鼠血清抗体测定(OD450 士SD) A conventional ELISA method for measuring the serum antibody titer assay results in Table 2. Table immunized mouse serum antibody 2IgE microspheres (Shi of OD450 SD)

Figure CN102008719AD00131

[0154] 结果显示:C ε 3肽-微球免疫小鼠抗体滴度大于30000。 [0154] The results show: C ε 3 peptide - antibody titers in mice immunized with microsphere is greater than 30,000. 免疫微球效果优于现行常规方法:肽与BSA化学交联法,即较C ε 3肽-BSA组滴度高10倍以上。 Current conventional immune microspheres better than: peptide to BSA chemical cross-linking, i.e. more groups C ε 3 -BSA peptide titers than 10 times higher.

[0155] 实施例9表皮生长因子受体III型突变体(EGFRvIII)微球 [0155] Example 9 EGFR type III mutant (EGFRvlll) microspheres embodiment

[0156] 一、包裹牛血清白蛋白(BSA+OVA)的PLGA/壳聚糖微球制备同实施例2。 [0156] a wrapped bovine serum albumin (BSA + OVA) in PLGA / chitosan microspheres prepared in Example 2.

[0157] 二、参考以下文献委托公司化学合成EGFRvIII表位多肽 [0157] Second, the reference to the literature commissioned chemically synthesized polypeptide epitope EGFRvIII

[0158]序列为:CGADSYEMEEDGVRKC [0158] sequence: CGADSYEMEEDGVRKC

[0159] 其合成方法参考文献(United States Patent : Antibodies to EGF receptor epitope peptides Johns, etal.7, 767,792August3, 2010) [0159] The synthesis method reference (United States Patent: Antibodies to EGF receptor epitope peptides Johns, etal.7, 767,792August3, 2010)

[0160] [0161] [0160] [0161]

分子。 molecule. [0162] [0162]

[0163] [0163]

[0164] [0164]

[0165] [0165]

三、微球与抗原多肽的连接用常规戊二醛方法,见实例4 Third, the antigenic polypeptide connection microspheres glutaraldehyde by conventional methods, see Example 4

四、多肽与BSA偶联委托公司制备。 Fourth, the polypeptide conjugated to BSA prepared commissioned. 据公司提供数据每个BSA连接多肽在3.5 According to data provided by each of the polypeptide in connection BSA 3.5

实施例10表皮生长因子受体III型突变体(EGFRvIII)微球的动物试验结果一、动物试验分组 Animal test results of Example 10 EGFR type III mutant (EGFRvlll) a microsphere, animal testing packet

28只昆明种小鼠分四组,每组七只。 28 Kunming mice were divided into four groups of seven. 分别以下疫苗及相应对照免疫。 The following vaccines and corresponding controls were immunized. 1、PLGA微球-EGFR肽10 %微球100微升尾静脉注射免疫小鼠[0166] 2、EGFR肽20 μ g EGFR多肽与Friend佐剂混合,皮下多点注射 1, 100 [mu] l 10% microsphere immunized mice intravenously injected peptides -EGFR PLGA microspheres [0166] 2, EGFR adjuvant peptide and 20 μ g EGFR polypeptide Friend, subcutaneous injection at multiple sites

[0167] 3、BSA-EGFR肽30 μ g BSA-EGFR肽与Friend佐剂混合,皮下多点注射 [0167] 3, BSA-EGFR peptide 30 μ adjuvant BSA-EGFR peptide Friend g, subcutaneous injection at multiple sites

[0168] 4、未经任何处理的小鼠为空白对照 [0168] 4 without any blank control treated mice

[0169] 初次免疲后二周,加强免疫,以后每周一次共六周。 [0169] After the initial two weeks avoid fatigue, strengthen immunity, after once a week for six weeks. 最后一次免疫后10日后小鼠处死,眼球采血,分离血清。 After the last immunization the mice were sacrificed 10 days, eye blood serum was separated.

[0170] 二、抗体滴度测定:ELISA常规方法 [0170] Second, the antibody titer assay: ELISA conventional methods

[0171] 1、被测抗原分别以0.05MPH9.6碳酸缓冲液配制20ug/ml包被液,0.1ml每孔过 [0171] 1, respectively 0.05MPH9.6 test antigen formulated carbonate buffer 20ug / ml were coated overnight, 0.1ml per well

夜包被酶标板。 Night-coated microtiter plates.

[0172] 2、PBST洗涤3次,每孔以0.3ml无蛋白封闭液室温封闭2h。 [0172] 2, washed with PBST 3 times, 0.3ml per well blocking solution at room temperature protein free blocked 2h.

[0173] 3、洗涤3次分别加入梯度稀释的小鼠血清各0.1ml,37°C孵育2h。 [0173] 3, washed three times with serially diluted mouse sera were added to each 0.1ml, 37 ° C incubation 2h.

[0174] 4、PBST洗涤5次加入1 : 2000兔抗鼠-HRP每孔0.1ml,室温孵育lh。 [0174] 4, was washed with PBST 5 times with 1: 2000 rabbit anti-mouse -HRP each well 0.1ml, incubated at room temperature lh. 洗涤5次后分别加入O.lmlTMB显色,显色15min各加入50ul IM硫酸 After 5 washes were added O.lmlTMB color, color 15min sulfuric acid was added to each 50ul IM

[0175] 三、抗体测定结果如表3 [0175] Third, the antibody assay results shown in Table 3

[0176] 表3EGFR微球免疫小鼠血清抗体测定(OD450 士SD) [0176] Table 3EGFR serum antibody assay microsphere immunized mice (Shi of OD450 SD)

[0177] [0177]

Figure CN102008719AD00141

[0178] 结果显示,免疫微球-EGFRvIII肽免疫小鼠,血清抗体滴度大于30000。 [0178] The results show that immunization of mice immunized microspheres -EGFRvIII peptide, serum antibody titers of greater than 30,000. 比常规BSA方法高5倍以上。 Higher than the conventional method described above 5 times BSA. 为进一步研究提供了实验基础。 It provides an experimental basis for further research.

[0179] 实例1 lHer2琼脂微球疫苗构建及动物试验结果 Construction [0179] Example 1 lHer2 agar microsphere vaccine and animal test results

[0180] 一、抗原:参照文献合成Her2表位多肽。 [0180] I. Antigen: Synthesis of reference literature Her2 polypeptide epitope. 序列为:CQMWAPQWGPDC。 Sequence: CQMWAPQWGPDC. 其合成方法可参考文献(Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2/neul.The Journal of Immunology, 2004,173 : 394-401. Angelika B.Riemer, Markus Klinger, Stefan Wagner, Astrid Bernhaus) The synthesis method can Reference (Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2 / neul.The Journal of Immunology, 2004,173:. 394-401 Angelika B.Riemer, Markus Klinger, Stefan Wagner , Astrid Bernhaus)

[0181] 二、微球:琼脂糖/壳聚糖微球制备见实例3 [0181] Second, the microspheres: Agarose / chitosan microspheres prepared as described in Example 3

[0182] 三、包裹有BSA及OBV作为Th蛋白的琼脂糖微球通过壳聚糖氨基偶朕Her2肽 [0182] III, and wrapped with BSA as OBV agarose beads by chitosan amino Th protein Her2 peptide coupling I

[0183] 多肽与微球连接按实例4操作。 [0183] polypeptide microspheres connection operation in Example 4.

[0184] 四、多肽与BSA偶联用常规方法戊二醛连接方法,委托公司操作。 [0184] Fourth, the polypeptide conjugated to BSA by glutaraldehyde method of connecting a conventional method, commissioned operation. 据公司提供数据,每个BSA分子偶联多肽为4.0 According to data provided by each BSA molecule conjugated polypeptide is 4.0

[0185] 五、动物试验 [0185] Fifth, animal testing

[0186] 28只昆明种小鼠分四组,每组七只。 [0186] 28 Kunming mice were divided into four groups of seven. 分别以下疫苗及相应对照免疫。 The following vaccines and corresponding controls were immunized.

[0187] 1、微球-Her2 10 %免疫微球0.Iml尾经脉注射 [0187] 1, microspheres -Her2 10% microsphere immunized 0.Iml tail injection meridians

[0188] 2、Her2 20ygHer2肽与Friend佐剂混合,皮下多点注射 [0188] 2, Her2 20ygHer2 adjuvant peptide Friend, subcutaneous injection at multiple sites

[0189] 3、BSA-Her2 30ygBSA-Her2肽与Friend佐剂混合,皮下多点注射 [0189] 3, adjuvants BSA-Her2 30ygBSA-Her2 peptide Friend, subcutaneous injection at multiple sites

[0190] 4、未经任何处理小鼠 [0190] 4, mice without any treatment

[0191] 初次免疫后二周加强免疫。 [0191] the first time two weeks after the booster immunization. 每周免疫一次,共六周。 Immune once a week, for 6 weeks. 最后一次免疫10日后小鼠处死,眼球采血,分离血清。 The last immunization the mice were sacrificed 10 days, eye blood serum was separated.

[0192] 六、ELISA检测抗体 [0192] VI, ELISA detection antibody

[0193] 1、被测抗原分别以0.05MPH9.6碳酸缓冲液配制20ug/ml包被液,0.1ml每孔过 [0193] 1, respectively 0.05MPH9.6 test antigen formulated carbonate buffer 20ug / ml were coated overnight, 0.1ml per well

夜包被酶标板。 Night-coated microtiter plates.

[0194] 2、PBST洗涤3次,每孔以0.3ml无蛋白封闭液室温封闭2h。 [0194] 2, washed with PBST 3 times, 0.3ml per well blocking solution at room temperature protein free blocked 2h.

[0195] 3、洗涤3次分别加入梯度稀释的小鼠血清各0.1ml,37°C孵育2h。 [0195] 3, washed three times with serially diluted mouse sera were added to each 0.1ml, 37 ° C incubation 2h.

[0196] 4、PBST洗涤5次加入1 : 2000兔抗鼠-HRP每孔0.1ml,室温孵育lh。 [0196] 4, was washed with PBST 5 times with 1: 2000 rabbit anti-mouse -HRP each well 0.1ml, incubated at room temperature lh. 洗涤5次后分别加入O.lmlTMB显色,显色15min各加入50ul IM硫酸 After 5 washes were added O.lmlTMB color, color 15min sulfuric acid was added to each 50ul IM

[0197] 七、实验结果:见表4 [0197] Seven experimental results: Table 4

[0198] 表4Her2微球免疫小鼠血清抗体测定(OD450 士SD) [0198] Table 4Her2 immunized mice serum antibody assay microspheres (Shi of OD450 SD)

[0199] [0199]

Figure CN102008719AD00151

[0200] 结果显示,免疫微球_Her2肽免疫小鼠获得高于3200滴度抗体。 [0200] The results show that immunization of mice immunized microspheres obtained _Her2 peptide antibody titer higher than 3200. BSA-Her2方法高5倍以上。 BSA-Her2 method of more than 5 times higher.

Claims (10)

1. 一种克服B细胞免疫耐受的免疫微球,其包括微球介质,其特征在于,所述微球介质外部包覆有疫苗抗原,所述微球介质内部包裹有用于活化T细胞的免疫载体蛋白。 An immune tolerance against B-cell immune microspheres, the microspheres comprising a medium, wherein said external medium microspheres coated with a vaccine antigen, the microspheres internal wrapping medium for activated T cells immunogenic carrier protein.
2.如权利要求1所述的克服B细胞免疫耐受的免疫微球,其特征在于,所述疫苗抗原的B细胞表位位于所述免疫微球的表面,所述免疫载体蛋白的T细胞表位位于所述免疫微球的内部,从而实现B细胞表位与T细胞表位的物理隔离。 2. B cell tolerance against immune microspheres according to claim 1, wherein said B cell epitope vaccine antigens located on the surface of the microspheres immunized, the carrier protein immune T cells immune epitope is located inside the microspheres to achieve physical isolation and B cell epitopes of the T cell epitope.
3.如权利要求1所述的克服B细胞免疫耐受的免疫微球,其特征在于,所述的免疫载体蛋白选自:嘁血蛋白、牛血清白蛋白、卵白蛋白、基因工程表达的破伤风病毒蛋白片断、乙肝病毒壳蛋白、脑膜炎球菌蛋白、HIV外壳蛋白、感冒病毒蛋白。 3 against the B cell tolerance by the immune microspheres according to claim 1, wherein said immunogenic carrier protein is selected from: breaking limpet albumin, bovine serum albumin, ovalbumin, expression of the genetically engineered cold virus protein fragment, hepatitis B virus capsid protein, meningococcal protein, HIV coat protein, influenza virus proteins.
4.如权利要求1所述的克服B细胞免疫耐受的免疫微球,其特征在于,所述的疫苗抗原选自:表皮生长因子、血管生长因子、血管生长因子受体、IgE、表皮生长因子受体EGFR及突变体、表皮生长因子受体肿瘤坏死因子、转化生长因子血管紧张素、CD20。 4. B cell tolerance against immune microspheres according to claim 1, wherein the vaccine antigen is selected from: epidermal growth factor, vascular endothelial growth factor, vascular endothelial growth factor receptor, IgE, epidermal growth and mutant factor receptor (EGFR), epidermal growth factor receptor tumor necrosis factor, transforming growth factor angiotensin, CD20.
5.如权利要求1所述的克服B细胞免疫耐受的免疫微球,其特征在于,所述微球介质选自:海藻酸钙、聚乳酸、聚乳酸-乙醇酸、琼脂糖、葡聚糖、蛋白。 5 against the B cell tolerance by the immune microspheres according to claim 1, wherein said medium is selected from the microspheres: calcium alginate, polylactic acid, polylactic acid - glycolic acid, agarose, dextran sugar, protein.
6.如权利要求1所述的克服B细胞免疫耐受的免疫微球,其特征在于,所述疫苗抗原通过常规化学方法与所述微球介质偶联。 6. B cell immunity against the immune tolerance of the microspheres as claimed in claim 1, wherein said vaccine antigen by conventional chemical methods coupled with the microspheres medium.
7.如权利要求6所述的克服B细胞免疫耐受的免疫微球,其特征在于,利用双功能交联试剂实现氨基-氨基、羧基-氨基、氨基-锍基的偶联。 7. immune tolerance against B-cell immune microspheres according to claim 6, characterized in that, using a bifunctional crosslinking reagent to achieve amino - amino group, a carboxyl group - amino, - coupling sulfonium group.
8.如权利要求7所述的克服B细胞免疫耐受的免疫微球,其特征在于,利用戊二醛试剂实现氨基与氨基的偶联。 Against B cell tolerance by the immune microspheres as claimed in claim 7, characterized in that, to achieve glutaraldehyde coupling agent and an amino group of an amino group.
9.如权利要求1-8中任一项所述的克服B细胞免疫耐受的免疫微球在疫苗制备上的应用。 9. Application of any of claims 1-8 B cell tolerance by the immune microspheres in the preparation of a vaccine according to overcome.
10.如权利要求1-8中任一项所述的克服B细胞免疫耐受的免疫微球在免疫佐剂上的应用。 10. Application of any of claims 1-8 B cell tolerance by the immune microspheres on one of the immunoadjuvant overcome.
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