牛Nramp1基因N端單克隆抗體的制備及鑒定
發(fā)布時間：2018-09-07 16:33
【摘要】：天然免疫力相關巨噬細胞蛋白1(Natural resistance-associated macrophage protein 1,Nramp1),是一種在有吞噬功能細胞(如巨噬細胞、噬中性粒細胞)中特異性表達的蛋白,具有二價金屬離子轉運功能。Nramp1定位于晚期胞內體上,主要通過將滿足病原微生物自身營養(yǎng)代謝或合成防御體系所必須的金屬離子(如Fe~(2+)、Mn~(2+))從吞噬體內轉運到胞液中而實現(xiàn)對病原微生物的抑制,從而增強機體抵御胞內病原微生物的能力。為了檢測Nramp1的轉基因細胞、胚胎,培育抗胞內病原微生物(如結核桿菌、布魯氏桿菌等)的轉基因牛及后續(xù)對轉基因牛的檢測鑒定。我們制備了牛Nramp1-N單克隆抗體(McAb)。 本試驗結果如下: 1篩選了誘導表達Nramp1-N蛋白的最佳培養(yǎng)條件,結果證明:在35℃搖床培養(yǎng)條件下,IPTG的終濃度為0.1 mmol/L,誘導時間為10 h時,Nramp1-N蛋白的表達量相對最大。使用AKTA蛋白純化系統(tǒng)得到純度較高的目的蛋白。 2用純化的Nramp1-N蛋白免疫Balb/c小鼠和家兔,采用間接ELISA方法來檢測免疫后小鼠和家兔的血清抗體效價,小鼠的血清抗體效價超過1∶1×10~5,而兔的血清抗體效價達到1∶1×10~6。 3融合骨髓瘤細胞和免疫脾細胞后,采用間接ELISA法檢測陽性克隆孔,檢測結果:細胞克隆率達到29.2 %,陽性細胞克隆率達到46.4 %。使用有限稀釋法克隆篩選后得到了兩株陽性雜交瘤細胞,并分別命名為D3和F6。采用ELISA和Western Blot方法對McAb進行特異性鑒定,結果證明所獲得的兩株細胞株所分泌的抗體均是針對Nramp1-N蛋白的單克隆抗體。使用秋水仙素對雜交瘤細胞進行核型分析,通過觀察100個中期雜交瘤細胞,統(tǒng)計后每個雜交瘤細胞染色體平均數(shù)目53±2對。 4應用間接ELISA方法檢測所獲得的細胞株培養(yǎng)上清和腹水的效價,D3和F6細胞培養(yǎng)上清效價分別為1:500和1:200;小鼠腹水效價分別為1:1×10~5和1:1×10~4。經反復凍存和傳代培養(yǎng)后,證明其均具有穩(wěn)定分泌抗體的能力。D3和F6所分泌的McAb的親和常數(shù)分別為2×10~8,2×10~7。D3和F6細胞株分泌的抗體分別屬于IgG1/κ、IgM/κ類型。
[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，
本文編號：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，
本文編號：2228805
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