产气荚膜梭菌β毒素单克隆抗体的制备及鉴定

发布时间：2018-08-15 19:48

【摘要】：产气荚膜梭菌(C.perfringens)又名魏氏梭菌是一种广泛分布于自然界和动物肠道中的细菌,属于条件性致病菌。可产生多种外毒素和酶类,其主要毒素是α、β、ε和ι,其中β毒素具有较强的神经毒性、细胞毒性和致死性。能够引起羊猝狙,犊牛、羔羊、仔猪肠毒血症,以及人和动物坏死性肠炎。在家畜感染造成快速的发病死亡,因此,称其为‘家畜猝死症’。在多种畜禽和野生动物发病,尤其3周内的幼小动物更易感染,给我国畜牧业造成了严重的损失。在80年代中期,我国曾经广泛而严重的爆发流行,后因抗生素的使用,使得该病的流行受到一定程度的抑制,有时临床症状不明显,但是仍有大量的报道。农业部将其列为二类传染病。本研究的主要目的是利用较纯的产气荚膜梭菌β毒素制备单克隆抗体。B、C型产气荚膜梭菌产生β毒素主要致死性毒素,但是C型菌也产生少量的α毒素。本研究首先采用了SephadexG-25对粗提的产气荚膜梭菌外毒素进行除盐,然后又通过SephadexG-200对浓缩液进行提纯的方法,最后得到了相对较纯的β毒素。我们将纯化的β毒素通过聚丙烯酰胺凝胶电泳(SDS-PAGE)对分离的蛋白纯度进行检测。所以采用该方法纯化的蛋白对单克隆抗体的制备起了很重要的作用。本研究采用了天然纯化的β毒素对6-8周龄的BALB/c小鼠进行免疫,加强免疫3天后取免疫小鼠脾脏细胞与骨髓瘤细胞Sp2/0进行细胞融合。经过间接ELISA筛选和有限稀释亚克隆法获得了2株稳定分泌针对C型产气荚膜梭菌β毒素的阳性的杂交瘤细胞株。将生长状态良好的阳性杂交瘤细胞注入小鼠腹腔内,待小鼠腹部膨大后收集腹水。采用正辛酸-硫酸铵纯化方法进行纯化腹水,从而获得产气荚膜梭菌β毒素的单克隆抗体。本实验成功获得了两株抗β毒素的杂交瘤细胞,分别命名为1G7、2F4。对所得到的两株抗β毒素的杂交瘤细胞分泌的抗体经过间接ELISA方法可知,小鼠腹水中单抗的效价显著高于杂交瘤细胞上清的抗体效价,最高可达到1:102400。经过Western Blot分析可知两种单克隆抗体均能与天然纯化的β毒素发生特异性反应,而与其他的毒素不发生反应。单克隆抗体具有生物活性单一、纯度高、与抗原结合的特异性强等优点,我们可以用单克隆抗体很快的检测疾病及治疗疾病。进而将制备的抗β毒素单克隆抗体通过聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western-blot)实验进一步验证了抗β毒素单克隆抗体的纯度和反应原性。为下一步快速构建有效检测产气荚膜梭菌毒素类型的检测技术方法奠定了基础。该方法在疫病快速检测、食品安全等方面具备很好的应用前景。
[Abstract]:Clostridium perfringens (C.perfringens), also known as Clostridium Wei, is a kind of bacteria widely distributed in nature and animal intestines. The main toxins are 伪, 尾, 蔚 and l, among which 尾 toxin has strong neurotoxicity, cytotoxicity and lethality. Can cause sudden amnion, calves, lambs, piglets, enterotoxemia, and necrotizing enteritis in humans and animals. Infection in domestic animals causes rapid morbidity and death, so it is called'sudden death in domestic animals'. In many animals and wild animals, especially the young animals within 3 weeks are more susceptible to infection, which has caused serious losses to animal husbandry in China. In the middle of 1980s, there was a widespread and serious outbreak of epidemic in our country. Later, the prevalence of the disease was restrained to a certain extent due to the use of antibiotics, sometimes the clinical symptoms were not obvious, but there were still a lot of reports. The Ministry of Agriculture classified it as a Class II infectious disease. The main purpose of this study was to prepare monoclonal antibody, Clostridium perfringens type C. perfringens 尾 -toxin by using pure Clostridium perfringens 尾 toxin, but a small amount of 伪 toxin was also produced by Clostridium perfringens. In this study, the crude extract of Clostridium perfringens exotoxin was desalted by SephadexG-25, then purified by SephadexG-200, and a relatively pure 尾 toxin was obtained. The purified 尾-toxin was detected by polyacrylamide gel electrophoresis (SDS-PAGE). So the protein purified by this method plays an important role in the preparation of monoclonal antibody. In this study, natural purified 尾 -toxin was used to immunize 6-8 week old BALB/c mice. After 3 days of enhanced immunization, the spleen cells of the immunized mice were fused with myeloma cell Sp2/0 for cell fusion. Two hybridoma cell lines secreting 尾 -toxin against Clostridium perfringens type C were obtained by indirect ELISA screening and limited dilution subcloning. The positive hybridoma cells in good growth state were injected into the abdominal cavity of mice and ascites were collected after abdominal enlargement. A monoclonal antibody against 尾 -toxin of Clostridium perfringens was obtained by purification of ascites with n-octanoic acid-ammonium sulfate. In this study, two hybridoma cells were successfully obtained, named as 1G7G 2F 4. By indirect ELISA method, the titer of monoclonal antibody in mouse ascites was significantly higher than that in the supernatant of hybridoma cells, and the highest titer was 1: 102400. Western Blot analysis showed that both monoclonal antibodies could react specifically with natural purified 尾 -toxin, but not with other toxins. Monoclonal antibodies have the advantages of single biological activity, high purity and strong specificity of binding with antigens, so we can quickly detect diseases and treat diseases with monoclonal antibodies. The purity and reactivity of the monoclonal antibody against 尾 -toxin were further verified by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western-blot). It lays a foundation for the rapid construction of a rapid detection method for the detection of Clostridium perfringens toxin types. The method has a good prospect in rapid detection of epidemic disease and food safety.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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