【作者】 徐晓彬；

【导师】 靳亚平；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 硕士

【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Fe²⁺、Mn²⁺)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∶1×10⁵,而兔的血清抗体效价达到1∶1×10⁶。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1:1×10⁵和1:1×10⁴。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2×10⁸,2×10⁷。D3和F6细胞株分泌的抗体分别属于IgG1/κ、IgM/κ类型。更多还原

【Abstract】 Natural resistance-associated macrophage protein 1（Nramp1）,is an iron transporter expressed exclusively within phagocytosis cells （such as macrophages and neutrophils）. Nramp1 was found to localize to late endosomes, where it mediates the export of iron and manganese into the cytosol. It has been proposed that Nramp1 promotes host resistance by depleting the phagolysosome of divalent metals(such as Fe²⁺and Mn²⁺), which are essential for pathogen growth. It enhances the organism resists ability of intracellular pathogenic microorganisms. In order to detect transgenic cells, embryos and transgenic cloned cattle of carrying Nramp1 and breed transgenic cattle that resist intracellular pathogens （such as Mycobacterium tuberculosis, Brucellosis, etc）. We prepared bovine Nramp1-N monoclonal antibody （McAb）.The experimental results are as follows:1 We have sieved the best way of expressing Nramp1-N Fusion Protein by induction of IPTG, Shaked cultivation at 35℃, Final concentration of IPTG: 0.1 mmol/L, the time: 10 h. We obtain Nramp1-N Fusion Protein that was higher purity by AKTA protein purified systerm.2 Using indirect ELISA to detect the titer of antibody in serum. The titer of antibody in mouse and rabbit immunized by Nramp1-N Fusion Protein reach up to 1:1×10⁵ and 1:1×10⁶.3 After fusion myeloma cells and immune spleen cells, using indirect ELISA to detect masc-clone, Cloning efficiency: 29.2 %, Masc-Cloning efficiency: 46.4 %. Subclone the positive cells by limiting dilution for several times to get identical cells, we got two positive hybridoma cells named D3 and F6. Using ELISA and Western Blot to detect specificity of two McAbs. The results showed two McAbs is aim at Nramp1-N. Using Colchcine to karyotype analysis of the two hybridoma cells, The average number of chromosome of the hybridoma cell lines was 53±2.4 The ELISA titer for bovine Nramp1-N antibodies in culture supernant was 1:500 and 1:200; The ELISA titer in ascites was 1:1×10⁵ and 1:1×10⁴. The antibody-serecting ability of the two hybridoma cells keep stable after serial subcultivation and resuscitation several times. The antibody-serecting ability of them keep stable after frozening and subcloning several times. Affinity constant of monoclonal Antibodys from D3 and F6 are 2×10⁸ and 2×10⁷. The Ig subtypes of D3 and F6 antibodies were IgG1/κand IgM/κ.更多还原