林麝源铜绿假单胞菌外膜蛋白的提取及免疫原性的研究

发布时间：2018-04-27 02:00

本文选题：铜绿假单胞菌 + 外膜蛋白　；参考：《四川农业大学》2015年硕士论文

【摘要】：铜绿假单胞杆菌一直以来是困扰野生动物养殖的一大难题,属较难防控的细菌性疾病之一。该菌具有较强的耐药性,感染机体后,可产生多种毒力因子,其具有很强的致病作用,单独使用抗生素的治疗效果并不理想,这限制了抗生素的广泛使用。因此,合理的疫苗接种对该疫病的预防起着至关重要的作用。目前,我国还没有针对铜绿假单胞菌病的有批文的疫苗。本研究根据林麝养殖场铜绿假单胞菌的分布情况,通过动物攻毒试验,成功筛选出了一株毒力最强的菌株作为试验菌株。经提取和纯化该菌株的外膜蛋白,并进行免疫接种,从细胞免疫、体液免疫和攻毒保护三个方面来评估该菌株的免疫原性,并对免疫效果进行评价,以期为林麝铜绿假单胞菌病的防控提供参考依据。本文所开展的研究和取得的结果如下：1.试验菌株的筛选试验选取本实验室从林麝体内分离并保存的7株铜绿假单胞菌,并均用同一菌液浓度对试验小鼠进行攻毒,从中筛选出一株毒力最强的菌株(sp-19株),最终,测得其对小鼠的LDso是6.7114×106 CFU。2.外膜蛋白的纯化及鉴定通过试剂盒提取sp-19株的外膜蛋白。经SDS-PAGE电泳分析,显示该方法提取的外膜蛋白有8个蛋白条带,其分子量分别约为104 KD、71 KD、52 KD、43 KD、36KD、 22KD、18 KD和9 KD。免疫印迹结果显示,这些外膜蛋白均能与抗铜绿假单胞菌sp-19株阳性血清发生特异性反应,具有良好的反应原性。3.外膜蛋白免疫效果评价分别将试剂盒提取的sp-19株纯化外膜蛋白、sp-19株全菌制备的免疫原和超声破碎提取的sp-19株外膜蛋白免疫实验小鼠。在四免后,采用间接ELISA的方法检测试验小鼠的抗体动态变化,结果显示,各抗原免疫组小鼠体内抗体滴度随免疫进程持续升高。其中,试剂盒提取的sp-19株纯化外膜蛋白抗原组小鼠的抗体水平最高,sp-19株全菌制备的免疫原组和超声破碎提取的sp-19株外膜蛋白抗原组次之,其效价分别达到1：12500、1：2500和1：500。体外淋巴细胞增殖试验表明,sp-19株外膜蛋白能显著促进各抗原免疫组的小鼠T、B淋巴细胞的增殖,其增殖率分别为3.05、2.51和1.33：采用细胞因子检测试剂盒对免疫0、15、29、43和50天各抗原免疫组小鼠血液中的IL-4和IFN-y进行检测,其含量最高达104pg/ml、87 pg/ml.66 pg/ml和134pg/ml、131 pg/ml和97 pg/ml。4.外膜蛋白免疫保护性研究分别用试剂盒提取的sp-19株纯化外膜蛋白、sp-19株全菌制备的免疫原和超声破碎提取的sp-19株外膜蛋白免疫小鼠,二免后用5倍LDso的sp-19株活菌经腹腔攻毒,结果显示,试剂盒提取的sp-19株纯化外膜蛋白保护率为87.5%,sp-19株全菌制备的免疫原保护率为62.5%,超声破碎提取的sp-19株外膜蛋白保护率仅为12.5%,生理盐水对照组和佐剂对照全部死亡。被动免疫试验中,先分别将各抗原组免疫56天后的高免血清0.2 ml腹腔注射于小鼠体内,2h后用2倍LDso的铜绿假单胞菌sp-19株活菌经腹腔攻毒。结果显示,试剂盒提取的sp-19株纯化外膜蛋白保护率为100%,全菌制备的免疫原保护率为87.5%,超声破碎提取的sp-19株外膜蛋白为仅25%,生理盐水对照组全部死亡。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) has been a difficult problem in wild animal breeding. It is one of the most difficult and resistant bacterial diseases. It has strong resistance. After infection, it can produce a variety of virulence factors. It has a strong pathogenic effect. The treatment effect of antibiotics alone is not ideal, which restricts the widespread of antibiotics. Therefore, a reasonable vaccination plays a vital role in the prevention of the disease. At present, there is no vaccine against Pseudomonas aeruginosa in China. According to the distribution of Pseudomonas aeruginosa in the forest musk farm, a strain with the strongest virulence has been successfully screened by the animal attack test. The outer membrane protein of the strain was extracted and purified, and the immunization was carried out. The immunogenicity of the strain was evaluated from three aspects of cell immunity, humoral immunity and attack of poison, and the immune effect was evaluated in order to provide reference for the prevention and control of Pseudomonas aeruginosa in forest musk deer. The results are as follows: 1. the screening test of the test strain selected 7 strains of Pseudomonas aeruginosa isolated and preserved from the musk deer in our laboratory, and used the concentration of the same bacteria to attack the test mice and screened out a strain of the strongest virulence (sp-19 strain). Finally, it was found that the LDso of the mice was 6.7114 * 106 CFU.2. outer membrane protein. The outer membrane protein of sp-19 strain was extracted and identified by the kit. The SDS-PAGE electrophoresis analysis showed that the extraction of the outer membrane protein had 8 protein bands, and its molecular weight was about 104 KD, 71 KD, 52 KD, 43 KD, 36KD, 22KD, 18 KD and 9 KD. showed that all these outer membrane proteins could be positive with the sp-19 strain of Pseudomonas aeruginosa. The specific response of the serum has a good response to the reactivity of the.3. outer membrane protein. The immunogenicity of the sp-19 strain, the immunogen prepared by the sp-19 strain and the outer membrane protein of the sp-19 strain extracted by the ultrasonic fragmentation of the immune test mice, was evaluated respectively. The indirect ELISA method was used to detect the experimental mice after four immunization. The antibody dynamic changes showed that the antibody titer of the mice in the antigen immunization group increased with the immune process. The antibody level of the sp-19 strain extracted by the kit was the highest, the immunogen prepared by the sp-19 strain and the outer membrane protein antigen of the sp-19 strain extracted by the ultrasonic fragmentation were the group. The proliferation test of 1:12500,1:2500 and 1:500. in vitro showed that the outer membrane protein of sp-19 strain could significantly promote the proliferation of T and B lymphocytes in the antigen immunized mice, and the proliferation rate was 3.05,2.51 and 1.33, respectively. The cytokine detection kit was used to immunize 0,15,29,43 and I in the blood of each antigen immunized group of 50 days. L-4 and IFN-y were tested with the highest content of 104pg/ml, 87 pg/ml.66 pg/ml and 134pg/ml, 131 pg/ml and 97 pg/ml.4. outer membrane proteins, the immune protective study of the outer membrane protein extracted with the reagent box, the immunogen prepared by the sp-19 strain and the sp-19 strain of the ultrasonic breakage of the outer membrane protein, was immunized with the 5 times LDso. The results showed that the protective rate of the purified outer membrane protein extracted from the sp-19 strain of the kit was 87.5%, the immunogen protection rate of the sp-19 strain was 62.5%, the protection rate of the outer membrane protein of the sp-19 strain extracted by ultrasonic fragmentation was only 12.5%, and the normal saline control group and the adjuvant control were all dead. Don't intraperitoneally injecting high serum free serum 0.2 ml from each antigen group for 56 days. After 2h, the sp-19 strain of Pseudomonas aeruginosa with 2 times LDso was used in the abdominal cavity. The results showed that the protection rate of the purified outer membrane protein extracted by the kit was 100%, the immune Yuan Bao protection rate of the whole bacteria was 87.5%, and the outer membrane of the sp-19 strain extracted by ultrasonic breakage was the outer membrane. The protein was only 25%, and all of the normal saline control group died.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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