牛Nramp1基因N端单克隆抗体的制备及鉴定
发布时间：2018-09-07 16:33
【摘要】：天然免疫力相关巨噬细胞蛋白1(Natural resistance-associated macrophage protein 1,Nramp1),是一种在有吞噬功能细胞(如巨噬细胞、噬中性粒细胞)中特异性表达的蛋白,具有二价金属离子转运功能。Nramp1定位于晚期胞内体上,主要通过将满足病原微生物自身营养代谢或合成防御体系所必须的金属离子(如Fe~(2+)、Mn~(2+))从吞噬体内转运到胞液中而实现对病原微生物的抑制,从而增强机体抵御胞内病原微生物的能力。为了检测Nramp1的转基因细胞、胚胎,培育抗胞内病原微生物(如结核杆菌、布鲁氏杆菌等)的转基因牛及后续对转基因牛的检测鉴定。我们制备了牛Nramp1-N单克隆抗体(McAb)。 本试验结果如下: 1筛选了诱导表达Nramp1-N蛋白的最佳培养条件,结果证明:在35℃摇床培养条件下,IPTG的终浓度为0.1 mmol/L,诱导时间为10 h时,Nramp1-N蛋白的表达量相对最大。使用AKTA蛋白纯化系统得到纯度较高的目的蛋白。 2用纯化的Nramp1-N蛋白免疫Balb/c小鼠和家兔,采用间接ELISA方法来检测免疫后小鼠和家兔的血清抗体效价,小鼠的血清抗体效价超过1∶1×10~5,而兔的血清抗体效价达到1∶1×10~6。 3融合骨髓瘤细胞和免疫脾细胞后,采用间接ELISA法检测阳性克隆孔,检测结果:细胞克隆率达到29.2 %,阳性细胞克隆率达到46.4 %。使用有限稀释法克隆筛选后得到了两株阳性杂交瘤细胞,并分别命名为D3和F6。采用ELISA和Western Blot方法对McAb进行特异性鉴定,结果证明所获得的两株细胞株所分泌的抗体均是针对Nramp1-N蛋白的单克隆抗体。使用秋水仙素对杂交瘤细胞进行核型分析,通过观察100个中期杂交瘤细胞,统计后每个杂交瘤细胞染色体平均数目53±2对。 4应用间接ELISA方法检测所获得的细胞株培养上清和腹水的效价,D3和F6细胞培养上清效价分别为1:500和1:200;小鼠腹水效价分别为1:1×10~5和1:1×10~4。经反复冻存和传代培养后,证明其均具有稳定分泌抗体的能力。D3和F6所分泌的McAb的亲和常数分别为2×10~8,2×10~7。D3和F6细胞株分泌的抗体分别属于IgG1/κ、IgM/κ类型。
[Abstract]:Natural immune-associated macrophage protein 1 (Natural resistance-associated macrophage protein 1) is a protein specifically expressed in phagocytic cells, such as macrophages and neutrophils, with bivalent metal ion transport function. Nramp1 is located in the late intracellular body. The inhibition of pathogenic microorganisms is mainly achieved by transferring metal ions (such as Fe~ (2) MN ~ (2) from the phagocytic body to the cytosol, which are necessary to satisfy the nutritional metabolism or synthetic defense system of pathogenic microorganisms. Thus enhance the ability of the body to resist intracellular pathogenic microorganisms. In order to detect the transgenic cells and embryos of Nramp1, to cultivate transgenic cattle resistant to intracellular pathogenic microorganisms (such as Mycobacterium tuberculosis, Brucella, etc.), and to detect and identify the transgenic cattle. We prepared bovine Nramp1-N monoclonal antibody (McAb). The results were as follows: 1 the optimal culture conditions for inducing and expressing Nramp1-N protein were screened. The results showed that the expression of Nramp1-N protein was the largest when the final concentration of Nramp1-N was 0.1 mmol/L, and the induction time was 10 h under the condition of shaking table culture at 35 鈩，
本文编号：2228805
[Abstract]:Natural immune-associated macrophage protein 1 (Natural resistance-associated macrophage protein 1) is a protein specifically expressed in phagocytic cells, such as macrophages and neutrophils, with bivalent metal ion transport function. Nramp1 is located in the late intracellular body. The inhibition of pathogenic microorganisms is mainly achieved by transferring metal ions (such as Fe~ (2) MN ~ (2) from the phagocytic body to the cytosol, which are necessary to satisfy the nutritional metabolism or synthetic defense system of pathogenic microorganisms. Thus enhance the ability of the body to resist intracellular pathogenic microorganisms. In order to detect the transgenic cells and embryos of Nramp1, to cultivate transgenic cattle resistant to intracellular pathogenic microorganisms (such as Mycobacterium tuberculosis, Brucella, etc.), and to detect and identify the transgenic cattle. We prepared bovine Nramp1-N monoclonal antibody (McAb). The results were as follows: 1 the optimal culture conditions for inducing and expressing Nramp1-N protein were screened. The results showed that the expression of Nramp1-N protein was the largest when the final concentration of Nramp1-N was 0.1 mmol/L, and the induction time was 10 h under the condition of shaking table culture at 35 鈩，
本文编号：2228805
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