【摘要】：產(chǎn)氣莢膜梭菌(C.perfringens)又名魏氏梭菌是一種廣泛分布于自然界和動(dòng)物腸道中的細(xì)菌,屬于條件性致病菌。可產(chǎn)生多種外毒素和酶類(lèi),其主要毒素是α、β、ε和ι,其中β毒素具有較強(qiáng)的神經(jīng)毒性、細(xì)胞毒性和致死性。能夠引起羊猝狙,犢牛、羔羊、仔豬腸毒血癥,以及人和動(dòng)物壞死性腸炎。在家畜感染造成快速的發(fā)病死亡,因此,稱(chēng)其為‘家畜猝死癥’。在多種畜禽和野生動(dòng)物發(fā)病,尤其3周內(nèi)的幼小動(dòng)物更易感染,給我國(guó)畜牧業(yè)造成了嚴(yán)重的損失。在80年代中期,我國(guó)曾經(jīng)廣泛而嚴(yán)重的爆發(fā)流行,后因抗生素的使用,使得該病的流行受到一定程度的抑制,有時(shí)臨床癥狀不明顯,但是仍有大量的報(bào)道。農(nóng)業(yè)部將其列為二類(lèi)傳染病。本研究的主要目的是利用較純的產(chǎn)氣莢膜梭菌β毒素制備單克隆抗體。B、C型產(chǎn)氣莢膜梭菌產(chǎn)生β毒素主要致死性毒素,但是C型菌也產(chǎn)生少量的α毒素。本研究首先采用了SephadexG-25對(duì)粗提的產(chǎn)氣莢膜梭菌外毒素進(jìn)行除鹽,然后又通過(guò)SephadexG-200對(duì)濃縮液進(jìn)行提純的方法,最后得到了相對(duì)較純的β毒素。我們將純化的β毒素通過(guò)聚丙烯酰胺凝膠電泳(SDS-PAGE)對(duì)分離的蛋白純度進(jìn)行檢測(cè)。所以采用該方法純化的蛋白對(duì)單克隆抗體的制備起了很重要的作用。本研究采用了天然純化的β毒素對(duì)6-8周齡的BALB/c小鼠進(jìn)行免疫,加強(qiáng)免疫3天后取免疫小鼠脾臟細(xì)胞與骨髓瘤細(xì)胞Sp2/0進(jìn)行細(xì)胞融合。經(jīng)過(guò)間接ELISA篩選和有限稀釋亞克隆法獲得了2株穩(wěn)定分泌針對(duì)C型產(chǎn)氣莢膜梭菌β毒素的陽(yáng)性的雜交瘤細(xì)胞株。將生長(zhǎng)狀態(tài)良好的陽(yáng)性雜交瘤細(xì)胞注入小鼠腹腔內(nèi),待小鼠腹部膨大后收集腹水。采用正辛酸-硫酸銨純化方法進(jìn)行純化腹水,從而獲得產(chǎn)氣莢膜梭菌β毒素的單克隆抗體。本實(shí)驗(yàn)成功獲得了兩株抗β毒素的雜交瘤細(xì)胞,分別命名為1G7、2F4。對(duì)所得到的兩株抗β毒素的雜交瘤細(xì)胞分泌的抗體經(jīng)過(guò)間接ELISA方法可知,小鼠腹水中單抗的效價(jià)顯著高于雜交瘤細(xì)胞上清的抗體效價(jià),最高可達(dá)到1:102400。經(jīng)過(guò)Western Blot分析可知兩種單克隆抗體均能與天然純化的β毒素發(fā)生特異性反應(yīng),而與其他的毒素不發(fā)生反應(yīng)。單克隆抗體具有生物活性單一、純度高、與抗原結(jié)合的特異性強(qiáng)等優(yōu)點(diǎn),我們可以用單克隆抗體很快的檢測(cè)疾病及治療疾病。進(jìn)而將制備的抗β毒素單克隆抗體通過(guò)聚丙烯酰胺凝膠電泳(SDS-PAGE)和免疫印跡(Western-blot)實(shí)驗(yàn)進(jìn)一步驗(yàn)證了抗β毒素單克隆抗體的純度和反應(yīng)原性。為下一步快速構(gòu)建有效檢測(cè)產(chǎn)氣莢膜梭菌毒素類(lèi)型的檢測(cè)技術(shù)方法奠定了基礎(chǔ)。該方法在疫病快速檢測(cè)、食品安全等方面具備很好的應(yīng)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 偽, 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 偽 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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S852.61

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