小鼠干扰素诱导蛋白IFIT1多克隆抗体的制备及应用

发布时间：2018-06-04 03:40

本文选题：IFIT1 + 多克隆抗体　；参考：《重庆医科大学》2007年硕士论文

【摘要】： 第一部分:小鼠干扰素诱导蛋白IFIT1多克隆抗体的制备目的:获取小鼠IFIT1 ( Interferon-induced protein with tetratricopetide repeats 1)特异性多克隆抗体,为深入研究IFIT1的结构和功能奠定基础。方法:扩增培养高效表达IFIT1的重组子pMAL-C2X-IFIT1,超声破碎后的细胞上清过Amylose Pre-packed column亲和层析柱,将所得纯化的融合蛋白MBP-IFIT1作为抗原,添加福氏佐机剂后免疫兔,收集抗血清,Western blot和ELISA方法测定抗血清特异性反应和效价。结果:纯化的MBP-IFIT1可诱导兔产生特异性免疫应答,所得抗体能够特异性识别融合蛋白中的IFIT1,ELISA结果显示其效价为1:12800。结论:MBP-IFIT1具有良好的抗原性,以该融合蛋白为抗原免疫兔成功制备出IFIT1特异性多克隆抗体。第二部分:小鼠多器官及细胞株干扰素诱导蛋白IFIT1的表达情况目的:观察小鼠多器官和多细胞株干扰素诱导蛋白IFIT1 RNA水平的表达情况。方法:将10只雄性正常小鼠C57/129小鼠断颈处死,取心、肝、脾、肺、肾、小肠、淋巴细胞和骨骼肌提RNA。用5μg/ml的内毒素(LPS)刺激Raw264.7细胞株、3T3细胞株和10T1/2细胞株5h后,终止刺激,收集细胞提RNA,同时设定正常对照组。将提纯的RNA分别进行RT-PCR,观察IFIT1的表达情况。结果:小鼠心、肝、脾、肺、肾、小肠、淋巴细胞和骨骼肌中均表达IFIT1,且存在表达量上的差别,心表达量最高,肝与小肠比较P=0.307,其它各组织间比较P0.05。Raw264.7细胞株、3T3细胞株和10T1/2细胞株LPS刺激5h后,均能诱导IFIT1表达,正常对照组表达为阴性。结论:IFIT1在小鼠体内为广泛表达,LPS刺激可多种细胞株表达IFIT1。第三部分:放射性损伤对小鼠干扰素诱导蛋白IFIT1及JAB1表达的影响目的:观察放射性损伤后早期小鼠肝组织IFIT1、JAB1各时相点变化特点,初步探讨创伤应激早期糖皮质激素抵抗的分子机制。方法:将20只C57小鼠,不拘雌雄,随机分为正常对照组和致伤组,致伤组采用5Gry 60Co一次性全身照射,分别于伤后1h、4h、和12h脱颈处死取肝组织。用western blot测定肝组织IFIT1、JAB1表达变化。结果:放射损伤后1h IFIT1开始增高,12h达最高,呈上升趋势,差异显著P0.01;同时,JAB1在1h开始下降,12h降到最低,呈下降趋势,差异显著P0.01。结论:放射损伤早期不仅引起小鼠肝组织IFIT1的显著增高,同时引起JAB1的显著下降,成相反趋势,揭示创伤应激早期糖皮质激素抵抗可能与IFIT1对其的阻遏作用和JAB1对其的正性调控有关。第四部分:颈交感神经阻滞对烧伤早期小鼠肝干扰素诱导蛋白IFIT1表达的影响目的:探讨颈交感神经阻滞(cervical sympathetic block,SB)对烧伤后早期小鼠肝脏干扰素诱导蛋白IFIT1表达的影响。方法:雄性C57/129小鼠50只,随机分为对照组和SB治疗组,TBSA 15-20%Ⅲ度小鼠烧伤模型,分别于烧伤前、烧伤后1h、6h、12h和24h取肝组织,采用免疫印迹法(Western blot)观察IFIT1的蛋白表达情况。结果:对照组烧伤后肝IFIT1表达显著增高(P0.01),烧伤后治疗组IFIT1表达量较对照组相应时相点显著降低(P0.01)。结论:烧伤后早期GR功能抑制可能与IFIT1负性调控有关,SB对烧伤的治疗作用可能是通过抑制IFIT1表达来实现的。
[Abstract]:Part one: preparation of mouse polyclonal antibody against interferon inducible protein IFIT1
Objective: to obtain the specific polyclonal antibody of mouse IFIT1 (Interferon-induced protein with tetratricopetide repeats 1), and to lay the foundation for the in-depth study of the structure and function of IFIT1.
Methods: the recombinant IFIT1 recombinant pMAL-C2X-IFIT1 was amplified and cultured, and the cells after ultrasonic fragmentation were cleared by Amylose Pre-packed column affinity chromatography column. The purified fusion protein MBP-IFIT1 was used as antigen, and the rabbit was immunized with FFL, and antiserum was collected, Western blot and ELISA methods were used to determine the specific reaction of antiserum and the antiserum. Titer.
Results: the purified MBP-IFIT1 can induce a specific immune response in rabbits. The antibody can specifically identify the IFIT1 in the fusion protein, and the ELISA results show that its titer is 1:12800..
Conclusion: MBP-IFIT1 has good antigenicity. IFIT1 specific polyclonal antibody was successfully prepared by immunizing rabbits with the fusion protein as antigen.
The second part: the expression of interferon induced protein IFIT1 in multiple organs and cell lines in mice: To observe the expression of interferon induced protein IFIT1 RNA in multiple organs and multicell lines in mice.
Methods: 10 male normal mice were killed in C57/129 mice, the heart, the liver, the spleen, the spleen, the lung, the kidney, the small intestine, the lymphocyte and the skeletal muscle were used to stimulate the Raw264.7 cell line, the 3T3 cell line and the 10T1/2 cell line 5h, and the 3T3 cell line and the 10T1/2 cell line 5h, to collect the cell extract RNA and set the normal control group. The purified RNA was carried out RT-PC, respectively. R, observe the expression of IFIT1.
Results: IFIT1 was expressed in the heart, liver, spleen, spleen, lung, kidney, kidney, kidney, small intestine, lymphocyte and skeletal muscle, and there was a difference in expression, the expression of heart was the highest, the liver and small intestine were compared with P=0.307, the other tissues were compared with P0.05.Raw264.7 cell lines, 3T3 cell lines and 10T1/2 fine cell LPS stimulated 5h, and the expression of the normal control group was expressed as the normal control group. Negative.
Conclusion: IFIT1 is widely expressed in mice, and LPS can stimulate IFIT1. expression in many cell lines.
The third part: the effect of radiation injury on the expression of interferon inducible protein IFIT1 and JAB1 in mice.
Objective: To observe the changes of the phase points of IFIT1 and JAB1 in early mice liver tissue after radiation injury, and to investigate the molecular mechanism of glucocorticoid resistance at the early stage of trauma stress.
Methods: 20 C57 mice were randomly divided into normal control group and injury group, which were randomly divided into normal control group and injury group. The injured group was irradiated by 5Gry 60Co in one time. The liver tissues were killed after 1h, 4h, and 12h removed respectively. The expression of IFIT1 and JAB1 in liver tissue was measured with Western blot.
Results: after radiation injury, 1H IFIT1 began to increase, and 12h reached the highest, showing an upward trend, and the difference was significant P0.01. At the same time, JAB1 began to decline in 1H, and the 12h decreased to the lowest level, and the difference was significant P0.01..
Conclusion: early radiation injury not only caused a significant increase in the IFIT1 of liver tissue in mice, but also caused a significant decrease in JAB1. It was suggested that the early glucocorticoid resistance of traumatic stress may be related to the repression of IFIT1 and the positive regulation of JAB1 on it.
The fourth part: the effect of cervical sympathetic blockade on the expression of interferon inducible protein IFIT1 in the early stage of burn injury in mice.
Objective: To investigate the effect of cervical sympathetic block (SB) on the expression of interferon induced protein IFIT1 in the liver of mice after burn. Methods: 50 male C57/129 mice were randomly divided into control group and SB treatment group, TBSA 15-20% III degree mice burn model, before burn, 1H, 6h, 12h, and liver tissue after burn, respectively. Western blotting (Western blot) was used to observe the protein expression of IFIT1.
Results: the expression of IFIT1 in the control group was significantly increased (P0.01), and the expression of IFIT1 in the treatment group was significantly lower than that of the control group (P0.01). Conclusion: the inhibition of GR function in the early stage of burn may be related to the negative regulation of IFIT1. The therapeutic effect of SB on burn may be achieved by inhibiting the expression of IFIT1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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