CN1559607A - 一种核酸物质与蛋白质类组合物及其生产方法和在免疫调节中的应用 - Google Patents
一种核酸物质与蛋白质类组合物及其生产方法和在免疫调节中的应用 Download PDFInfo
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Abstract
一种抑制细胞免疫反应的核酸物质与蛋白质类的免疫组合物及其生产方法和使用方法,其组合物含有核酸物质和蛋白质或含有核酸物质与多肽或含有核酸物质与灭活病原体和或含有核酸物质与含矿物油的灭活疫苗的组合物;其制备方法是:将核酸物质与蛋白质类抗原物质进行混合。其使用方法是:通过注射、喷射、口服、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法使上述组合物进入机体。本发明的技术效果:利用本发明提供的组合物及生产和使用方法不仅使核酸物质与蛋白质或多肽或灭活病原体为抗原组合物有效的抑制特异性的细胞免疫反应能力,而且副作用低,制备简单无需复杂设备和操作简单,并且易于实施,其免疫抑制效果超过未使用本发明组合物。
Description
一、技术领域
本发明涉及一种核酸物质与蛋白质类组合物及其生产方法和其在制备免疫制剂中的应用,特别是用DNA与蛋白质或灭活的微生物体混合而生成的核酸与蛋白质的组合物及其生产方法和在免疫调节中的应用。
二、背景技术
机体的免疫系统始终处在一种复杂调节过程中。免疫调节是指免疫应答过程中免疫系统内部各细胞之间、免疫细胞与免疫分子之间以及免疫系统与其他系统之间的相互作用,构成一个相互协调、相互制约的网络结构,使免疫应答维持合适的强度,从而保证机体内环境的稳定。在外来病原物入侵后,免疫系统可以根据病原物的特点激活需要的免疫反应来抵御和清除病原物。免疫反应又分为体液免疫反应和细胞免疫反应。其中体液免疫反应是以产生特异性抗体的反应,而细胞免疫反应是激活T细胞为主的免疫反应。提高机体免疫力的最主要的手段是接种疫苗。目前已有多种方法用来生产抗传染性病原体的疫苗,如灭活疫苗,减毒活疫苗、重组疫苗,亚单位疫苗和DNA疫苗等,它们的基本作用原理是相同的,即借助病原体的抗原物质在体内被免疫细胞识别而激发免疫反应,达到免疫个体不被传染性病原体感染的目的。但是,机体免疫力过强,也会产生副作用,如自身免疫疾病等。所以在外来抗原物质入侵时,机体会利用一整套免疫调节机制平衡免疫反应。人为的抑制免疫反应可是用于治疗自身免疫疾病的手段之一。T细胞免疫抑制是机体免疫功能的一个重要环节,比如:限制自身免疫疾病发生和机能性下调免疫反应。T细胞可以以通过无共刺激分子存在情况下、通过T细胞为APC细胞刺激下或最近证明的胸腺来源的CD4+CD25+细胞与新生T细胞相互作用来实现。多数自身免疫疾病均存在特异性的抗原受体,如系统红斑狼疮病人血液中,临床检查发现存在着抗DNA抗体,这种抗全与抗原形成了免疫复合物,沉淀在组织中引起周期性的炎症。又如类风湿病(RA)在患者的关节组织中,含有自身免疫反应性T-细胞,它能与某种未知抗原发生反应,这类T细胞通过T细胞抗原受体(TCRs)不仅可识别特定的抗原,也可识别主要组织相溶性分子(MHC)。因此,自身免疫反应的抗原受体通过早期识别,引发炎症,导致临床上系统红斑狼疮,类风湿关节炎等严重的自身免疫性疾病。
实验研究已确定了某些自身免疫疾病的抗原受体,如小鼠和大鼠动物模型中发生的NEB/NEW小鼠的红斑狼疮,实验性接种髓磷脂碱性蛋白(MBP),过敏性脑脊髓炎(EAE)。
在小鼠红班狼疮中,采用抗特异性抗体(anti-Ids)去除产生自身免疫反应的B细胞,达到治疗的目的,部分临床病例表明抗特异性抗体能明显缓病症(B.Hahn 1983),但有些病例表明抗特异性抗体使病情恶化(R.Schwartz 1983)。同样在脑脊髓炎的治疗中,利用免疫TCR-衍生肽类对抗自身免疫反应的TCRs,结果有些病情得到了缓解,有些病情则进一步恶化(Heber-Katz 1990)。
因此,利用免疫接种来治疗某些自身免疫疾病时,病人的免疫反应直接影响治疗的临床效果。若免疫接种导致产生抗体反应并形成抗Ids抗体,这些抗Ids可能会结合自身免疫反应中的B细胞或T细胞,引起体外调节性的溶解反应,达到临床上病情缓解的效果,相反,如果免疫反应导致体内清除抗-Ids,免疫反应物就会和自身免疫反应中的B-细胞或T-细胞结合,同时又交叉性地与它们的抗原受体结合,进一步激发免疫细胞产生更多的自身免疫反应抗体(Abs)或T-细胞,使临床病情恶化,免疫接种激发的大多数T-细胞,使临床病情恶化,免疫接种激发的大我数T-细胞反应,会同时引起辅助T-细胞反应病情恶化或杀伤抑制细胞反应病情缓解。因此,有效地治疗自身免疫疾病的免疫学方法尚需深入研究。
在临床上通常使用的免疫抑制剂和方法有如下几个方面:利用化学药品如:普乐可复(FK506),环孢素A(CsA),骁悉(MMF),硫唑嘌呤(Aza),强的松(Pred),早基强的松龙(MP);利用抗体如:抗淋巴细胞球蛋白(ALG),抗CD4单克隆抗体(OKT4);但以上的免疫抑制剂都有其毒副作用,若使用不当,一方面可因过度抑制机体免疫反应性而引发多种并发症,另一方面也可因其自身的毒副作用导致抑制器官功能衰竭。
三、发明内容
本发明的目的是针对现有免疫抑制技术存在的上述问题,提供一种可以用于制备各种免疫抑制制剂的核酸类和蛋白质类组合物及其制备方法,其特征在于:使用DNA片段或质粒与蛋白质类物质或与灭活的病原体抗原物质通过简单混合后生成的组合物直接注入机体,而产生其机体的免疫抑制反应。利用本发明组合物制备的抑制机体免疫力的免疫制剂,不仅可以降低副作用,而且可以有效的降低和抑制机体的细胞为主的免疫反应。
本发明核酸物质和蛋白质类组合物是由DNA片段或DNA质粒与蛋白质或与灭活的病原体抗原物质通过简单混合后生成的组合物,具有如下通式:
DNA+蛋白质,或DNA+灭活病原体抗原
一种核酸物质和蛋白质类组合物,其特征在于该组合物含有DNA片段或DNA质粒与蛋白质或DNA和灭活病原体抗原物质的组合物。
上述DNA是人工合成的或者通过从微生物、真核及植物细胞或组织提取而来的双链DNA或质粒。
上述的蛋白质类为人工合成的或者通过生物体生产而成的蛋白质或多肽或病原体经公知的方法灭活后的抗原物质。
上述生物体为大肠杆菌或芽孢杆菌或酵母菌或真核细胞等在人工培养条件下可用于生产放大的生物体。
上述的灭活病原体通过生物体分离和生产的病毒、菌、寄生虫及过敏物质经公知的方法灭活后而得到的无感染性病原体,其特征在于该灭活病原体直接与DNA混合或灭活病原体经与矿物油乳化后再与DNA混合。
上述抑制免疫反应的脱氧核糖核酸和蛋白质类物质的组合物通过以下方法得到:将DNA与蛋白质或DNA与灭活病原体抗原物质溶于生理盐水中混合后,制得本发明的组合物。其中DNA与蛋白质或DNA与灭活病原体抗原的质量比值为0.1∶1~1∶10。
上述抑制免疫反应的DNA与含矿物油的灭活疫苗的组合物通过以下方法得到:将DNA溶于生理盐水中后再溶于含矿物油的灭活疫苗乳剂中混合后,制备本发明的组合物。其中DNA与含矿物油的灭活疫苗的质量比值为0.1∶1~1∶10。
上述抑制细胞免疫反应的核酸物质与蛋白质类的免疫组合物的使用方法是:通过注射、喷射、口服、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法使上述组合物进入机体。
通过试验,本发明人发现:本发明的组合物可激发机体产生正常的特异性抗体免疫反应,但是抑制特异性细胞免疫反应,特别是Th1型免疫反应。试验已经证明使用本发明组合物的免疫方法产生的特异性细胞免疫抑制反应是通过增强白介素10水平和抑制伽马干扰素(IFN-γ)水平介导的。增强白介素10水平是机体免疫系统抑制过强的免疫反应而进行的有效调节作用。防止机体中不必要的免疫损伤而重要手段之一。本发明正是利用了这一调节作用,发现了使用本发明的核酸类和蛋白质类组合物及免疫机体后能有效地抑制特异性细胞反应,并可广泛地应用于制备各种免疫抑制制剂。
本发明与现有技术相比具有以下优点:
1、本发明组合物比化学药品如:普乐可复(FK506),环孢素A(CsA),骁悉(MMF),硫唑嘌呤(Aza),强的松(Pred),早基强的松龙(MP),和抗体如:OKT4等更为安全,有选择性的抑制机体T细胞免疫反应,从而可以有效的应用于治疗自身免疫疾病、器官移植、控制T细胞水平的治疗等方面;
2、本发明组合物能够有效地激发机体特异性的体液免疫反应,抑制机体特异性的T细胞免疫反应,所以可以特异性地用于抑制特定病原物引起的免疫损伤。而本发明组合物能有效地克服免疫抑制方法不特异的不足;
3、本发明组合物不要求特殊的反应条件,一般药厂的普通设备即可生产,生产方法简单,提纯容易,易于工业化生产;
4、本发明组合物用途广泛,可以用于致病病原体有:病毒、原核细胞、真核细胞,真核细胞病原体包括细胞致病病原体和多细胞寄生虫类。
5、本发明组合物用途广泛,可以用于自身免疫反应病原有:系统红斑狼疮、类风湿关节炎、慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天龅疮、类天龅疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性多发性多神经炎等严重的自身免疫性疾病。
6、本发明组合物用途广泛,可以用于器官移植中的免疫排斥反应。
四、附图说明
图1.SuperY/VP1重组质粒的酶切鉴定
用限制性内切酶EcoR I和Xba I酶切口蹄疫VP1 cDNA产物,纯化回收后,将VP1基因片段克隆入穿梭质粒SuperY。
A:PCR扩增FMDV vp1基因的电泳结果;B:重组质粒SuperY/VP1的酶切鉴定.
泳道1和3:DNA Marker;泳道2:VP1基因片段;泳道4:SuperY/VP1经EcoRI/Xba I酶切后的结果。从图中可以看出,进行双酶切(EcoR I/Xba I)鉴定,得到到泳道4下方639bp左右有一条带,证明VP1已克隆至SuperY上,命名为SuperY/VP1。经序列分析验证无误。VP1基因成功克隆于SuperY质粒中。
图2.SuperY/VP1重组质粒表达产物的SDS-PAGE
样品加热变性后,经SDS-PAGE电泳并卡马氏亮兰G250染色。
泳道1:酵母SMD1168上清;泳道2:SuperY的酵母表达上清;泳道3:SuperY/VP1的酵母表达上清;泳道4:低分子量蛋白标准。从图中可以看出,SuperY/VP1中的VP1基因在酵母细胞中表达。
图3.重组质粒VP1表达产物的Western blotting分析。
样品加热变性后,经SDS-PAGE分离蛋白质,电转移至硝酸纤维素膜,再于牛抗口蹄疫高免血清和二抗反应后,显色。
泳道1:低分子量蛋白标准;泳道2:SuperY/VP1的酵母表达上清;泳道3:酵母SMD1168上清;泳道4:SuperY的酵母表达上清。
泳道2在66kD和43kD附近出现了特异性的显色带,而其它泳道中均未出现条带,表明表达的重组蛋白能与抗FMDV血清特异性反应带,证明表达产物具有FMDV的免疫原性。
图4.比较DNA与146s的样品混合后性质的变化
为证明核酸物质与蛋白类物质在混合后无性质上的改变,将DNA与146S抗原混合后置于37℃孵育24小时,经琼脂糖电泳,比较变化情况。图中显示,泳道1和2是未经混合的DNA样品,3和4是混合的DNA与146s的样品在电泳后的情况,泳道5、6是混合的DNA和146s样品经37℃孵育24小时后的情况。结果说明混合的DNA与146s的样品混合前后无变化。
图5.比较DNA与蛋白质或DNA与灭活病毒抗原组合后后免疫小鼠产生抗体滴度变化及影响:
为验证DNA与蛋白质或DNA与灭活病毒抗原组合物免疫小鼠后对免疫系统中抗体免疫水平的影响,在小白鼠肌肉注射本发明提到各种组合物后,比较免疫效果,在第二周再重复注射同等剂量各种组合物一次,每两周后取取血清测定其抗体变化结果如图。
从图中可以看出,当DNA与蛋白质VP1或DNA与灭活病毒抗原146S组合物免疫小鼠时,特异性抗体水平与其它组合物免疫后组比较无明显变化。说明其DNA与蛋白质或DNA与灭活病毒抗原组合后免疫动物,在激活特异性抗体水平是无特别变化。
图6.比较DNA与蛋白质组合后免疫小鼠对T细胞特异性扩增的影响:
为说明DNA与蛋白质VP1组合物组合物免疫小鼠后对免疫系统中细胞免疫水平的影响,在小白鼠肌肉注射本发明提到各种组合物后,比较免疫效果,在第二周再重复注射同等剂量各种组合物一次,两周后取脾脏T细胞测定其T细胞扩增活性,结果如图。
从图中可以看出,当DNA与蛋白质VP1组合物免疫小鼠时,T细胞扩增活性明显低于其它组合物免疫后的水平。说明其DNA与蛋白质组合后免疫动物,可以特异性的降低T细胞免疫水平。
图7.比较DNA与灭活病毒抗原或与灭活病毒油佐剂疫苗组合后免疫小鼠对T细胞特异性扩增的影响:
为说明DNA与灭活病毒抗原或DNA与灭活病毒油佐剂疫苗组合物免疫小鼠后对免疫系统中细胞免疫水平的影响,在小白鼠肌肉注射本发明提到各种组合物后,比较免疫效果,在第二周再重复注射同等剂量各种组合物一次,两周后取脾脏T细胞测定其T细胞扩增活性,结果如图。
从图中可以看出,当DNA与灭活病毒抗原146S或DNA与灭活病毒油佐剂疫苗组合物免疫小鼠时,T细胞扩增活性明显低于其它组合物免疫后的水平,其中DNA与灭活病毒油佐剂疫苗组合物免疫后降低的水平更多。说明其DNA与灭活病毒抗原或DNA与灭活病毒油佐剂疫苗组合后免疫动物,可以特异性的降低T细胞免疫水平。
图8.比较DNA与蛋白质或DNA与灭活病毒抗原组合物免疫后小鼠对细胞介素水平的影响
为说明DNA与蛋白质或DNA与灭活病毒抗原组合物影响免疫系统和细胞免疫水平,免疫后对小鼠的细胞介素IL-4、IL-10和IFN-γ进行检测。图中不同组合免疫后产生的细胞介素变化看出,当DNA与蛋白质或DNA与灭活病毒抗原组合物免疫小鼠时,IL-4和IL-10上升,而IL-2和IFN-γ下降。由此证明其组合物抑制T细胞活性是通过IL-4和IL-10水平完成的。
五、具体实施例
下面这些实施例是示范性的,而不是限制性的,可根据上述本发明的技术方案和实际情况,来确定具体的实施方式。下面的实施例可以帮助本领域的技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1 DNA的制备
一种方法是将动物组织低温后粉碎,在苯酚氯仿溶液中去掉蛋白质,双链DNA经乙醇沉淀而分离出来。
另一种方法是从植物组织中采用CTAB法提取DNA,在苯酚氯仿溶液中去掉蛋白质,双链DNA经乙醇沉淀而分离出来。
另一种方法是从大肠杆菌中提取质粒DNA,在苯酚氯仿溶液中去掉蛋白质,双链DNA经乙醇沉淀而分离出来。
以上提取方法和技术的详细描叙可在Sambrook等人的″Molecular Cloning″(第二版1998,Cold Spring Harbor Laboratory Press,纽约)和厉朝龙等编,《生物化学与分子生物学实验技术》浙江大学出版社查寻。
实施例2:蛋白质和多肽的制备
蛋白质和多肽可以通过标准的自动多肽合成仪(如ABI,433A等)合成,合成方法按仪器制造商使用方法;或从动物组织和细胞、植物组织和细胞或微生物中,按常规的蛋白质化学方法进行提取,也可以从基因工程表达菌或细胞中提取。这些多肽提取方法都是公知的,在Doonan的″Protein Purification Protocols″(1996,Humana Press,NJ)中有详细描叙。
实施例3:病原物的制备及灭活
病原体通过生物体分离和生产的病毒、菌、支原体、寄生虫及过敏物质经公知的灭活方法和试剂如:甲醛或福尔马林、β-丙内酯、N-乙酰乙烯亚胺以及二乙烯亚胺等,灭活后而得到的无感染性病原体,再经分离纯化后而制成。
实施例4:口蹄疫VP1 cDNA克隆及其表达的鉴定。
在我们申请的中国发明专利(公开号:1391954)中已有详细的描述。
实施例5:表达载体SuperY/VP1的构建与鉴定
用限制性内切酶EcoR I和Xba I酶切口蹄疫VP1 cDNA产物,经琼脂糖电泳,回收后,将VP1基因片段克隆入穿梭质粒SuperY的EcoR I和Xba I位点,转化至Top10 F′感受态细胞,筛选鉴定阳性克隆,并以序列分析鉴定。
实施例6:VP1在酵母中的表达与检测
将SuperY/VP1采用电击转化酵母,筛出单菌落30℃摇瓶培养48-96小时后,取上清进行SDS-PAGE电泳,考马斯亮蓝R-250染色,并用凝胶成像系统照相。VP1的表达产物分子量约66kD和43kD两种,将蛋白转移至NC膜,以5%脱脂牛奶作封闭剂,然后依次与牛抗口蹄疫高免血清、羊抗牛IgG-HRP酶标抗体孵育,最后在DAB/H2O2下显色。
实施例7:抗原的配制:
1、146S抗原的制备是由购自的兰州兽医研究所生产口蹄疫牛O型灭活疫苗,经去除灭活苗中的矿物油,保存在4℃。
2、生殖与呼吸综合症病毒(PRRSV)抗原是由购自的哈尔滨兽医研究所生产生殖与呼吸综合症病毒灭活疫苗,经去除灭活苗中的矿物油,保存在4℃。
3、纯化后的VP1蛋白质溶于0.9%生理盐水中,保存在4℃。
以上抗原,用Bradford法进行蛋白定量。
实施例8:测定核酸/蛋白质组合物实验:
为证明核酸物质与蛋白类物质在混合后无性质上的改变,将DNA与146S抗原混合后置于37℃孵育24小时,经琼脂糖电泳,比较变化情况。
图中显示,泳道1和2是未经混合的DNA样品,3和4是混合的DNA与146s的样品在电泳后的情况,泳道5、6是混合的DNA和146s的样品经37℃孵育24小时后的情况。
实施例9:小鼠免疫实验:
实验中用到的组合物均溶于0.9%NaCl中。将6-8周龄BALR/c(H-2d)雌性小鼠每组6只进行注射。每只小鼠肌肉多点注射100μg pcD-VP1重组质粒及相应的化学佐剂,第一次免疫后14天加强免疫一次。对照组注射空载体pcDNA3。分别在第0、35和50天收集血清。
实施例10:抗体的ELISA检测
在小白鼠肌肉注射100μg多核酸/肽/多聚物复合物后,在第二周再重复注射同等剂量一次,两周取血清对免疫血清进行抗体水平ELISA检测,测定其免疫反应。
将96孔酶标板用适量抗原包被,4℃过夜;弃去包被液PBST洗板三次;加入不低于1∶100稀释度的免疫动物血清,37℃孵育2小时;PBST洗板三次后,每孔加入适量辣根过氧化物酶标记的羊抗动物二抗(Ig)100μl 37℃孵育1小时后弃去;PBST洗三次,加入底物液100μl,室温显色反应30分钟,2M硫酸中止反应,用酶标仪测定相应波长的OD值。
实验结果见附图6。图中对照:pcDNA3,质粒:口蹄疫VP1 pcD-VP1质粒,组合物:为口蹄疫VP1多肽与pcD-VP1;口蹄疫146S抗原与pcD-VP1。
实施例11:T细胞扩增实验:
雌性BALB/c小鼠被分为6组。每一组分别注射重组质粒pcD-VP1 100μg与相应的化学佐剂。加强免疫后14天,无菌取脾制成单个细胞悬液[15]。用PBS液洗三次,离心并进行细胞计数,调整细胞浓度到1×106/ml,每组细胞悬液分4份加入96孔培养板中。其中一份加100μl的ConA至终浓度为5μg/ml,一份加146S抗原至终浓度为2μg/ml,一份加加100μl的BSA至终浓度为2μg/ml作为对照组,另一份不加刺激物,24h后,每孔加入100μl MTT至终浓度为5mg/ml,4h后,每孔加100μl SDS-DMSO(20%SDS溶于50%DMSO,pH2.0),使其完全溶解,在酶标仪上读取570nm处的OD值。
实施例12细胞介素水平测定
利用竞争定量PCR进行检测细胞介素mRNA的水平,其关键在于引入一个内标模板pQRS,它含有IL-2;IL;IL-5;IL-10;IL-12;TNF-α;TGF-β;IFN-γ;INOS;HRPT十个基因的部分序列,因此以pQRS可以检测鼠的相应细胞因子的含量。
取免疫后的小鼠断颈处死后取出脾脏,提取总RNA后,由RT-PCR扩增鼠脾脏总cDNA,其条件为:42℃反转录30-60分钟,99℃变性5分钟,5℃放置5分钟。PCR为:94℃,5分钟;94℃,40秒,55℃,20秒,72℃,40秒,扩增40个循环;72℃,10分钟。在1.5%琼脂糖电泳后观察结果。
Claims (12)
1、一种抑制细胞免疫反应的核酸物质与蛋白质类的免疫组合物,其特征在于该组合物含有核酸物质和蛋白质或含有核酸物质与多肽或或含有核酸物质与灭活病原体和或含有核酸物质与含矿物油的灭活疫苗的组合物。
2、如权利要求1所述的核酸物质,其特征在于该核酸物质为DNA片段或DNA质粒。
3、如权利要求1所述核酸物质是人工合成的或者通过从微生物或动物或植物细胞或组织提取而来的双链DNA或DNA质粒。
4、如权利要求1所述的蛋白质和多肽为利用公知的化学合成方法生产而得到的。
5、如权利要求1所述的蛋白质和多肽是通过公知的基因重组和基因工程的方法在生物体生产而得到的。
6、如权利要求1所述的灭活病原体通过生物体分离和生产的病毒、菌、寄生虫及过敏物质经公知的方法灭活后而得到的无感染性病原体,其特征在于该灭活病原体直接与DNA混合或灭活病原体经与矿物油乳化后再与DNA混合。
7、如权利要求5和6所述生物体为大肠杆菌或芽孢杆菌或酵母菌或真核细胞在人工培养条件下可用于生产放大的生物体。
8、如权利要求5和6所述生物体为动物体或植物体。
9、如权利要求1所述抑制免疫反应的DNA和蛋白质类物质的组合物通过以下方法得到:将DNA与蛋白质溶于生理盐水中混合后,制备本发明的组合物。其中DNA与蛋白质的质量比值为0.1∶1~1∶10。
10、如权利要求1所述抑制免疫反应的或DNA与灭活病原体抗原的组合物通过以下方法得到:将DNA与灭活病原体抗原溶于生理盐水中混合后,制备本发明的组合物。其中DNA与灭活病原体抗原的质量比值为0.1∶1~1∶10。
11、如权利要求1所述抑制免疫反应的DNA与含矿物油的灭活疫苗的组合物通过以下方法得到:将DNA溶于生理盐水中后再溶于含矿物油的灭活疫苗乳剂中混合后,制备本发明的组合物。其中DNA与含矿物油的灭活疫苗的质量比值为0.1∶1~1∶10。
12、如权利要求1所述的抑制细胞免疫反应的核酸物质与蛋白质类的免疫组合物的使用方法是:通过注射、喷射、口服、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法使上述组合物进入机体。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1909918B (zh) * | 2004-02-20 | 2012-08-22 | 中国农业大学 | T细胞免疫反应抑制剂 |
CN103239734A (zh) * | 2012-02-10 | 2013-08-14 | 北京艾棣维欣生物技术有限公司 | 用于预防和/或治疗呼吸道合胞病毒感染的疫苗 |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100374152C (zh) * | 2005-12-23 | 2008-03-12 | 中国农业大学 | 一种过敏性反应抑制剂 |
US8877206B2 (en) | 2007-03-22 | 2014-11-04 | Pds Biotechnology Corporation | Stimulation of an immune response by cationic lipids |
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PL2755680T3 (pl) * | 2011-09-12 | 2019-03-29 | Pds Biotechnology Corporation | Preparaty szczepionkowe w postaci cząstek |
CN103191442B (zh) * | 2012-01-04 | 2014-09-03 | 复旦大学 | 一种具有抗hiv-1病毒的结核基因疫苗及其制备方法和应用 |
TWI672149B (zh) | 2012-09-21 | 2019-09-21 | 美商Pds生技公司 | 改良之疫苗組成物及使用方法 |
JP2014028820A (ja) * | 2013-08-30 | 2014-02-13 | China Agricultural Univ | アレルギー抑制剤の組成物及びキットならびにその使用方法 |
CA3005251A1 (en) | 2015-11-13 | 2017-05-18 | Pds Biotechnology Corporation | Lipids as synthetic vectors to enhance antigen processing and presentation ex-vivo in dendritic cell therapy |
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CN1044092C (zh) * | 1997-02-26 | 1999-07-14 | 上海医科大学 | 抗原-抗体-重组dna复合型疫苗 |
US20060251667A1 (en) * | 2002-08-29 | 2006-11-09 | Chua Kaw Y | Recombinant nucleic acid useful for inducing protective immune response against allergens |
CN1559607A (zh) * | 2004-02-20 | 2005-01-05 | �й�ũҵ��ѧ | 一种核酸物质与蛋白质类组合物及其生产方法和在免疫调节中的应用 |
-
2004
- 2004-02-20 CN CNA2004100391891A patent/CN1559607A/zh active Pending
-
2005
- 2005-01-31 JP JP2006553418A patent/JP2007524688A/ja active Pending
- 2005-01-31 CA CA002556803A patent/CA2556803A1/en not_active Abandoned
- 2005-01-31 US US10/590,040 patent/US20070184037A1/en not_active Abandoned
- 2005-01-31 WO PCT/CN2005/000136 patent/WO2005079833A1/zh active Application Filing
- 2005-01-31 CN CN2005800027018A patent/CN1909918B/zh not_active Expired - Fee Related
- 2005-01-31 EP EP05706576A patent/EP1716863A4/en not_active Withdrawn
- 2005-01-31 AU AU2005215080A patent/AU2005215080A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1909918B (zh) * | 2004-02-20 | 2012-08-22 | 中国农业大学 | T细胞免疫反应抑制剂 |
CN103239734A (zh) * | 2012-02-10 | 2013-08-14 | 北京艾棣维欣生物技术有限公司 | 用于预防和/或治疗呼吸道合胞病毒感染的疫苗 |
Also Published As
Publication number | Publication date |
---|---|
JP2007524688A (ja) | 2007-08-30 |
CA2556803A1 (en) | 2005-09-01 |
US20070184037A1 (en) | 2007-08-09 |
CN1909918B (zh) | 2012-08-22 |
CN1909918A (zh) | 2007-02-07 |
AU2005215080A1 (en) | 2005-09-01 |
EP1716863A1 (en) | 2006-11-02 |
EP1716863A4 (en) | 2010-08-18 |
WO2005079833A1 (fr) | 2005-09-01 |
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