C、D型产气荚膜梭菌二价类毒素疫苗及抗毒素血清的研制

发布时间：2018-06-20 02:46

本文选题：C、D型产气荚膜梭菌 + 氢氧化铝佐剂　；参考：《山东农业大学》2015年硕士论文

【摘要】：产气荚膜梭菌在自然界是无处不在的,是多种健康动物肠道的常在菌,能造成人及多种动物发病,且有发病急、病程短、死亡快的特点,每年大量动物因感染产气荚膜梭菌而死亡,给养殖业造成巨大损失。C、D型产气荚膜梭菌能单独或混合感染牛羊等多种动物,引起动物坏死性肠炎及肠毒血症,且两型感染短时间内难以区分。为了控制产气荚膜梭菌引起的多种疾病,本试验研制了C、D型产气荚膜梭菌类毒素二价苗及抗毒素血清,有效预防并治疗C、D型产气荚膜梭菌病。随着抗生素的广泛应用,细菌耐药性逐渐增强,特别是产气荚膜梭菌病一旦发生,抗生素难以治疗,用产气荚膜梭菌疫苗预防是最佳选择。根据产气荚膜梭菌致病是由其所产生外毒素引起的致病机理,本试验研制了安全、高效的类毒素疫苗。疫苗效力往往与抗原浓度成正比,为了提高抗原浓度,本试验选取实验室最佳菌株,将C型菌与D型菌以4:3比例接种于同一培养基,高效产毒,测得毒素对小鼠的半数致死量2-5.8/mL,并用该毒素制备了产气荚膜梭菌铝胶苗和白油苗。我国现有产气荚膜梭菌商品苗均为灭活铝胶联苗,免疫后发病时有发生,因此本试验研制了产气荚膜梭菌二价苗。为了证明疫苗的安全性和有效性,将两种疫苗做安全性检验,测定外毒素对家兔的最小致死量,并使用不同剂量疫苗免疫家兔,21d后做攻毒保护试验。结果表明:两种疫苗免疫家兔,免疫部位均无溃疡及肉芽肿,28d后剖检免疫部位无残留疫苗存在,两种疫苗均安全性良好;C型毒素对家兔的最小致死量为0.04m L,D型毒素对家兔的最小致死量为0.4m L;两种疫苗不同剂量免疫家兔,通过攻毒保护试验,测得铝胶苗对家兔的最小免疫剂量为2mL,白油类毒素苗对家兔的最小免疫剂量为1mL。产气荚膜梭菌发病后用抗生素难以治疗。抗血清作为一种快速的特异性治疗制剂,能快速中和毒素,治疗由C、D型产气荚膜梭菌引起的中毒性疾病。本试验使用高浓度毒素制备的油苗作为第一免疫原,除菌外毒素作为第二免疫原,制备高效价抗毒素血清,用间接ELISA法监测抗体水平变化,并使用小鼠中和试验检验抗血清的抗体水平。结果表明:本试验制备的高效价C、D型产气荚膜梭菌抗毒素血清,每0.1mL血清能中和400个C型毒素对小鼠的MLD和600个D型毒素MLD,有良好的应用前景。
[Abstract]:Clostridium perfringens is ubiquitous in nature. It is a common bacteria in the intestinal tract of many healthy animals. It can make adults and many kinds of animals, and it has the characteristics of rapid onset, short course and fast death. A large number of animals die from the infection of Clostridium perfringens, causing huge loss of.C and D type Clostridium perfringens can be isolated or mixed. A variety of animals, such as cattle and sheep, can cause animal necrotizing enteritis and intestinal toxemia, and the type two infection is difficult to distinguish in a short time. In order to control a variety of diseases caused by Clostridium perfringens, this experiment has developed C, D type Clostridium perfringens toxoid and antitoxin serum, effective prevention and treatment of C, D type Clostridium perfringens disease. With resistance to resistance to Clostridium perfringens of type D. The antibiotic resistance of Clostridium perfringens is increasing, especially when Clostridium perfringens is occurring, antibiotics are difficult to treat, and Clostridium perfringens vaccine is the best choice. According to Clostridium perfringens, the pathogenic mechanism caused by the exotoxin produced by Clostridium perfringens is a safe and efficient vaccine. The potency is often proportional to the antigen concentration. In order to improve the antigen concentration, this experiment selects the best laboratory strain, inoculating C and D bacteria in the same medium with the same medium, producing the toxin efficiently and measuring half the lethal dose 2-5.8/mL of the mice, and using the toxin to prepare the aluminum and white oil production of Clostridium perfringens. In our country, the existing gas producing gas is produced. In order to prove the safety and effectiveness of the vaccine, the safety and effectiveness of the vaccine were tested and the minimum lethal dose of exotoxin to rabbits was measured in order to prove the safety and effectiveness of the vaccine, and the rabbits were immunized with different doses of vaccines and then attacked by 21d after 21d. The results showed that there were no ulcers and granulomatosis in the immune parts of the two vaccinated rabbits. After 28d, there was no residual vaccine in the immunization site, the two vaccines were all safe, the minimum lethal dose of C toxin to rabbits was 0.04m L, and the minimum lethal dose of D toxin to rabbits was 0.4m L; the two kinds of vaccines were immune to rabbits. The minimum immunization dose of aluminum gel seedlings to rabbits was 2mL, and the minimum immunization dose of white oil vaccine was 1mL. after the onset of Clostridium perfringens. The antiserum was a rapid and specific treatment preparation, which could quickly neutralize the toxin, and the treatment was caused by C and D type Clostridium perfringens. In this experiment, the oil vaccine prepared by high concentration toxin was used as the first immunogen, and the bacteria exotoxin was used as the second immunogen to prepare the high effective antitoxin serum. The antibody level was monitored by indirect ELISA, and the antibody level of the antiserum was tested by the neutralization test in mice. The results showed that the high effective price, C, D prepared by this experiment Clostridium perfringens antitoxin serum, which can neutralize 400 C toxin in each 0.1mL serum, has good application prospects for MLD and 600 D toxin MLD in mice.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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