本文關(guān)鍵詞： 氣腫疽梭菌 分離鑒定 cctA基因 原核表達(dá)　出處：《延邊大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：氣腫疽是由氣腫疽梭菌引起的一種急性、熱性、敗血性傳染病,因其具有發(fā)病快、死亡率高的特點(diǎn),給畜牧業(yè)的發(fā)展帶來了巨大的經(jīng)濟(jì)損失。該病主要以皮下和深部肌肉處發(fā)生氣性、炎性腫脹,按壓有捻發(fā)音為典型特征。而繼2006年之后吉林省延邊州敦化市、延吉市依蘭鎮(zhèn)、三道鎮(zhèn)相繼出現(xiàn)牛氣腫疽病例,這嚴(yán)重制約了延邊州乃至全國畜牧業(yè)的發(fā)展。隨著其易感群體的逐漸擴(kuò)大,氣腫疽梭菌也越來越多的威脅到了人類的安全。為了制備安全高效的新型氣腫疽疫苗,控制本地區(qū)氣腫疽病的發(fā)生,本研究對延邊州延吉市采集的疑似氣腫疽梭菌感染的牛肝臟、脾臟、肌肉等組織進(jìn)行了分離,應(yīng)用病原學(xué)檢查、生化鑒定、動物試驗(yàn)及分子生物學(xué)鑒定的方法對該菌進(jìn)行了鑒定。根據(jù)GenBank中已發(fā)表的氣腫疽梭菌cctA基因,設(shè)計合成一對特異性引物,并以分離純化的延邊株氣腫疽梭菌總DNA為模板擴(kuò)增出cctA基因,并進(jìn)行克隆載體的構(gòu)建。利用生物信息學(xué)方法,對cctA蛋白的理化性質(zhì)、結(jié)構(gòu)功能域、蛋白質(zhì)二級結(jié)構(gòu)和三級結(jié)構(gòu)及抗原表位等重要參數(shù)進(jìn)行了預(yù)測和分析。將克隆質(zhì)粒pMD 19-T-cctA進(jìn)行雙酶切后,與pEGX-4T-1原核表達(dá)載體進(jìn)行連接,構(gòu)建pGEX-4T-cctA原核表達(dá)重組質(zhì)粒,并對其陽性克隆誘導(dǎo)表達(dá),進(jìn)行SDS-PAGE和Western blot分析。研究結(jié)果表明：本試驗(yàn)成功的對延邊株氣腫疽梭菌進(jìn)行了分離,與參考序列(JQ692583)相比,共存在5處突變,核苷酸和氨基酸序列的同源性均為99.0%；該蛋白的理論等電點(diǎn)為6.79,不存在跨膜區(qū)和信號肽序列,二級結(jié)構(gòu)以p-折疊和無規(guī)則卷曲為主,主要有5個抗原表位區(qū)域。經(jīng)SDS-PAGE和Western blot分析顯示,可見約44 kDa大小的片段,并具有良好的反應(yīng)原性。此結(jié)果為延邊地區(qū)氣腫疽病的免疫和微生態(tài)防治提供了重要的參考依據(jù),并為CctA蛋白功能的深入研究提供了參考,并為氣腫疽梭菌單克隆抗體的制備及合成肽疫苗的研發(fā)奠定了一定的基礎(chǔ)。
[Abstract]:Emphysematous gangrene is an acute, caused by Clostridium chauvoei heat, septic infectious disease, which is due to the onset of fast, high mortality, brought huge economic losses to animal husbandry. The disease mainly in the subcutaneous and deep muscles at the occurrence of gas, inflammatory swelling, press twist pronunciation is typical. And since 2006 in Jilin Prefecture of Yanbian Province, Dunhua City, Yanji City, Yilan Town, three towns have appeared in bovine emphysema cases, which seriously restrict the development of Yanbian and the national animal husbandry. With gradual enlargement of the susceptible population, carbonis shuttle bacteria also more and more threatening the safety of people. In order to model blackleg vaccine preparation is safe and effective, control the local blackleg disease, the study of suspected Clostridium chauvoei infection of bovine liver, collected in Yanji Yanbian City, spleen, muscle and other tissues were isolated, disease. The original examination, biochemical test, animal test methods and molecular biological identification of the identification of the bacterium Clostridium chauvoei. According to the cctA gene published in GenBank, designed a pair of specific primers, and the separation and purification of Yanbian strains of Clostridium chauvoei total DNA by cctA gene as template expansion, and to construct the cloning vector. Using bioinformatics methods, physical and chemical properties of the cctA protein, protein structure and function domain, two level structure and three stage structure and important parameters of antigen epitopes were predicted and analyzed. The cloned plasmid pMD 19-T-cctA was digested, and the prokaryotic expression vector pEGX-4T-1 to connect construction of pGEX-4T-cctA Recombinant Prokaryotic expression plasmid, and the expression of SDS-PAGE positive clones, and Western blot analysis. The results show that: the successful test of the Yanbian strain of Clostridium chauvoei were separated, and Compared with the reference sequence (JQ692583), there were 5 mutation, nucleotide and amino acid sequence homology was 99%; the protein isoelectric point is 6.79. There is no transmembrane domain and signal peptide sequence, the two stage structure with p- sheet and coil, there are 5 main antigenic epitope regions by SDS-PAGE and Western. Blot analysis showed that the visible about 44 kDa fragments, and has a good immunogenicity. The results provide an important reference for the immune blackleg disease in Yanbian area and micro ecological control, and for further research of CctA protein function for reference, and makes some the foundation for the development and preparation of synthetic peptide vaccine against Clostridium chauvoei monoclonal antibody.

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 郭洪友;鄭聃;;牛氣腫疽病的診治[J];現(xiàn)代農(nóng)業(yè)科技;2010年07期

2 陸安法,徐官紅,簡廷安,姚振華;牛氣腫疽的防制[J];動物醫(yī)學(xué)進(jìn)展;2003年06期

3 趙小林;洪貴生;李榮蘭;;一起牛氣腫疽病的病例報道[J];中國牛業(yè)科學(xué);2014年01期

4 侯文軍,譚文宇;牛氣腫疽的診斷與防制[J];廣西畜牧獸醫(yī);2004年06期

5 陳長旺;;鄉(xiāng)村牛氣腫疽的調(diào)查及防治探討[J];廣西畜牧獸醫(yī);2010年01期

6 王鎖開;鐘為武;卓尚耀;熊恩培;彭紅;;一起牛氣腫疽流行病學(xué)調(diào)查[J];湖南畜牧獸醫(yī);2008年04期

7 梁宗炳;;襄樊市耕牛氣腫疽病調(diào)查報告[J];湖北畜牧獸醫(yī);1987年01期

8 王正興;;山區(qū)牛氣腫疽診斷與防制[J];上海畜牧獸醫(yī)通訊;2011年02期

相關(guān)碩士學(xué)位論文 前1條

1 車達(dá);氣腫疽梭菌單克隆抗體的制備及鑒定[D];延邊大學(xué);2011年

，

本文編號：1500456