本文選題：副豬嗜血桿菌 + 毒力因子　； 參考：《東北林業(yè)大學(xué)》2015年碩士論文

【摘要】：近年來副豬嗜血桿菌病呈爆發(fā)流行趨勢,死亡率高達(dá)50%,已逐漸成為世界范圍內(nèi)的重要細(xì)菌性疾病,給養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失。為了探討副豬嗜血桿菌(Haemophilus parasuis, HPS) HbpA蛋白的功能及其在致病中的作用,從新的角度詮釋HPS的毒力特性和感染致病機(jī)制,本研究應(yīng)用免疫蛋白質(zhì)組學(xué)技術(shù),以新發(fā)現(xiàn)的具有免疫原性的HbpA蛋白為切入點(diǎn),以功能基因組學(xué)為理論基礎(chǔ),從多方面多層次探索了HbpA蛋白生物學(xué)功能,研究結(jié)果如下：1、利用蛋白質(zhì)組學(xué)技術(shù)和技術(shù)篩選并鑒定得到HPS的一個(gè)具有免疫原性的周質(zhì)蛋白HbpA。2、利用基因重組技術(shù)構(gòu)建重組質(zhì)粒pET28a-HbpA并誘導(dǎo)表達(dá)出可溶性的目的蛋白,大小為59.4kD, PI=7.06。3、利用技術(shù)驗(yàn)證HbpA蛋白能和鼠源HbpA單抗發(fā)生特異性結(jié)合,證明了HbpA蛋白的特異性。4、利用Heme-agarose法驗(yàn)證了HbpA蛋白在體外環(huán)境下能Heme結(jié)合,當(dāng)HbpA的量一定時(shí),HbpA-Heme復(fù)合體的分子量在一定的范圍內(nèi)(0mM-4mM)隨Heme濃度的升高而增大；當(dāng)Heme濃度達(dá)到一定限度(4mM)后,HbpA-Heme復(fù)合體的分子量將不再增大,而是維持在一個(gè)恒定的數(shù)值。5、利用IEM技術(shù)確定野生株HbpA蛋白和Heme的復(fù)合體主要存在于細(xì)胞外膜,內(nèi)膜和細(xì)胞核。6、利用免疫組化技術(shù)直觀的證明了HPS在豬肺臟內(nèi)的確表達(dá)出HbpA蛋白。7、通過測定HPS的生長曲線證明了Fe離子是HPS生存、繁殖的重要元素。8、利用抗體體外殺菌技術(shù)證明了在體外環(huán)境下HbpA抗體能殺死近半數(shù)的HPS菌體。9、利用RT-PCR技術(shù)驗(yàn)證了HPS在Fe離子缺乏的環(huán)境下體內(nèi)HbpA基因表達(dá)量是正常培養(yǎng)時(shí)的2倍。本研究從分子水平闡明了HbpA蛋白在HPS的存活、繁殖和致病機(jī)制方面的功能,并從新的角度詮釋HPS的毒力特性和感染致病機(jī)制,對(duì)有效的疫苗和藥物阻斷劑的研究具有一定的理論和實(shí)踐意義。
[Abstract]:In recent years, Haemophilus parasuis disease has become an important bacterial disease in the world, which has caused huge economic losses to the pig industry. The mortality rate of Haemophilus paratosus is as high as 50%, and it has gradually become an important bacterial disease in the world. In order to investigate the function of Haemophilus parasuis, HPS) HbpA protein and its role in pathogenicity, the virulence of HPS and the pathogenesis of infection were interpreted from a new point of view. Based on the newly discovered immunogenicity HbpA protein and functional genomics, the biological function of HbpA protein was explored from many aspects and levels. The results are as follows: 1. A immunogenicity protein HbpA.2of HPS was screened and identified by proteomics techniques. The recombinant plasmid pET28a-HbpA was constructed by gene recombination technique and the soluble protein was induced to express. The size of HbpA protein was 59.4 kD and Pi was 7.06.3. The specific binding ability of HbpA protein to murine HbpA monoclonal antibody was verified by the technique, and the specificity of HbpA protein was proved by using Heme-agarose method. Heme-agarose method was used to verify that HbpA protein could bind to Heme in vitro. When the amount of HbpA is constant, the molecular weight of HbpA-Heme complex increases with the increase of Heme concentration in a certain range, and the molecular weight of HbpA-Heme complex will not increase when the concentration of Heme reaches a certain limit of 4mM). The IEM technique was used to determine that the complex of HbpA protein and Heme mainly existed in the extracellular membrane. The expression of HbpA protein. 7 in pig lung was proved by immunohistochemical technique. The growth curve of HPS showed that Fe ~ (2 +) was the survival of HPS. The important element of reproduction. 8. Using antibody bactericidal technique in vitro, it was proved that HbpA antibody could kill nearly half of HPS cell. RT-PCR technique was used to verify that the expression of HbpA gene in the environment of Fe ion deficiency was positive. Twice as much as in normal culture. The purpose of this study was to elucidate the function of HbpA protein in the survival, reproduction and pathogenesis of HPS at the molecular level, and to interpret the virulence and pathogenicity of HPS from a new perspective. It has certain theoretical and practical significance for the study of effective vaccines and drug blockers.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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相關(guān)期刊論文 前3條

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本文編號(hào)：1913660