本文選題：延邊地區(qū)　切入點(diǎn)：氣腫疽梭菌　出處：《延邊大學(xué)》2016年碩士論文

【摘要】：氣腫疽(Clostridium chauvoei infection),病原為氣腫疽梭菌,是一種急性、熱性、敗血性傳染病。常見(jiàn)的典型癥狀為肌肉豐滿(mǎn)部位發(fā)生炎性、氣性腫脹,觸壓有捻發(fā)音,有一定的地區(qū)流行性、季節(jié)性,多發(fā)生在潮濕的山谷牧場(chǎng)、天氣炎熱的多雨季節(jié)。近幾年吉林省延邊州相繼爆發(fā)牛氣腫疽,給畜牧業(yè)和養(yǎng)殖業(yè)的發(fā)展帶來(lái)了巨大的沖擊和經(jīng)濟(jì)損失,同時(shí)也逐漸地威脅到人類(lèi)的安全和生產(chǎn)。本研究根據(jù)NCBI上氣腫疽梭菌細(xì)胞毒素A(CctA)基因序列,設(shè)計(jì)兩對(duì)特異性引物。一方面,將采集延邊地區(qū)165頭份的牛血液樣本,分別進(jìn)行血涂片鏡檢和PCR檢測(cè),再應(yīng)用統(tǒng)計(jì)學(xué)對(duì)各組數(shù)據(jù)分析。另一方面,采用疑似感染氣腫疽梭菌的牛肝臟、脾臟、心臟及肌肉等組織進(jìn)行分離,應(yīng)用病原學(xué)檢查、生化鑒定、動(dòng)物實(shí)驗(yàn)、分子生物學(xué)方法進(jìn)行鑒定。將PCR擴(kuò)增的CctA基因克隆至pMD19-T simple載體,對(duì)測(cè)序正確的CctA基因序列分析,然后亞克隆至pVAX-1真核表達(dá)載體,將構(gòu)建的pVAXl-CctA真核表達(dá)質(zhì)粒轉(zhuǎn)入Vero細(xì)胞中,通過(guò)間接免疫熒光(IFAT)和免疫印跡(Western blotting)檢測(cè)CctA基因的表達(dá)產(chǎn)物。結(jié)果顯示,PCR方法檢測(cè)牛氣腫疽的平均陽(yáng)性率為15.2%,應(yīng)用統(tǒng)計(jì)學(xué)分析表明,牛氣腫疽感染率在不同縣市、不同品種之間差異性不顯著(P0.05)；而不同年齡、不同地理?xiàng)l件氣腫疽感染率差異性顯著(P0.05)；在個(gè)體農(nóng)戶(hù)的飼養(yǎng)方式中陽(yáng)性率顯著高于規(guī)模化的養(yǎng)殖方式(P0.01)；說(shuō)明牛感染氣腫疽梭菌主要受年齡段、地理?xiàng)l件、飼養(yǎng)方式等因素的影響較大。氣腫疽梭菌擴(kuò)增的CctA基因大小為501 bp,測(cè)序得到的CctA基因核苷酸與氨基酸序列與氣腫疽梭菌ATCC10092菌株進(jìn)行同源性比對(duì)分析,有3處點(diǎn)核苷酸突變,核苷酸序列同源性為99.4%；存在著2個(gè)位點(diǎn)的氨基酸特異性改變,氨基酸序列同源性為98.8%。轉(zhuǎn)染的pVAX1-CctA組的細(xì)胞有綠色熒光,且pVAX1-CctA重組質(zhì)粒表達(dá)產(chǎn)物大小約為19 kDa,表明pVAX1-CctA質(zhì)粒在Vero田胞中成功表達(dá)了CctA蛋白。本研究更好地預(yù)測(cè)和分析了本地區(qū)分離株的信息,對(duì)預(yù)防和控制氣腫疽提供了參考,為氣腫疽的核酸疫苗研制奠定了深厚的基礎(chǔ)。
[Abstract]:Clostridium chauvoei Infection.Clostridium chauvoei Infection.The pathogen is Clostridium chauvoei, which is an acute, hot and septic infectious disease. In recent years, cattle emphysema and gangrene have erupted in Yanbian Prefecture, Jilin Province, which has brought enormous impact and economic losses to the development of animal husbandry and aquaculture. In this study, two pairs of specific primers were designed according to the gene sequence of Clostridium emphysema cytotoxin (NCBI). On the one hand, 165 bovine blood samples were collected from Yanbian area. Blood smear examination and PCR examination were performed respectively, and then statistical analysis was used to analyze the data of each group. On the other hand, bovine liver, spleen, heart and muscle tissues suspected to be infected with Clostridium emphysema were isolated and pathologically examined. The CctA gene amplified by PCR was cloned into pMD19-T simple vector, the sequence of CctA gene was sequenced correctly, then subcloned into pVAX-1 eukaryotic expression vector. The constructed eukaryotic expression plasmid of pVAXl-CctA was transferred into Vero cells. The expression products of CctA gene were detected by indirect immunofluorescence (IFATT) and Western blotting. The results showed that the average positive rate of detection of bovine emphysema by PCR was 15.2g. The infection rate of emphysema in cattle was not significantly different among different species in different counties and cities, but in different ages. The difference of emphysema infection rate in different geographical conditions was significant (P 0.05), and the positive rate of emphysema infection rate in individual farmers was significantly higher than that in large-scale breeding method (P 0.01), which indicated that cattle infected with Clostridium emphysema were mainly affected by age and geographical conditions. The size of CctA gene amplified by C. emphysema was 501bp.The sequence of nucleotide and amino acid of CctA gene was compared with that of ATCC10092 strain of C. emphysema, and there were 3 point nucleotide mutations. The nucleotide sequence homology was 99.4, and there were two amino acid specific changes, and the amino acid sequence homology was 98.8. The transfected pVAX1-CctA cells had green fluorescence. The expression product size of pVAX1-CctA recombinant plasmid was about 19 kDa, which indicated that the CctA protein was successfully expressed by pVAX1-CctA plasmid in Vero field cells. The information of the isolated strains in the region was better predicted and analyzed in this study, which provided a reference for the prevention and control of emphysema. It lays a solid foundation for the development of nucleic acid vaccine for emphysema.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S858.23

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