本文選題：羊種布魯氏菌 + 巨噬細(xì)胞��； 參考：《中國疾病預(yù)防控制中心》2017年碩士論文

【摘要】：目的:建立布魯氏菌侵染巨噬細(xì)胞模型,比較4株羊種布魯氏菌變異株對巨噬細(xì)胞侵襲能力及胞內(nèi)生存能力,比較4株羊種布魯氏菌變異株的脂多糖特點(diǎn)及其遺傳學(xué)變化。方法:建立強(qiáng)毒菌株羊種布魯氏菌16M和弱毒菌株羊種布魯氏菌疫苗菌株M5侵染小鼠巨噬細(xì)胞RAW264.7細(xì)胞模型,以PBS處理組作為對照,用ELISA法測量侵染后4、8、24h細(xì)胞上清液中IL-1β、IL-6和IL-12的表達(dá)水平;通過涂平板計(jì)數(shù)測量侵染后2、4、8、24和48h巨噬細(xì)胞內(nèi)的布魯氏菌的數(shù)量變化,繪制布魯氏菌巨噬細(xì)胞內(nèi)生存曲線,比較羊種布魯氏菌強(qiáng)毒株16M和疫苗株M5在巨噬細(xì)胞內(nèi)存活能力的差異,建立布魯氏菌在巨噬細(xì)胞內(nèi)的生存曲線模型。用4株羊種布魯氏菌變異菌株2014186、2014035、2015031、2015033及犬種布魯氏菌RM6/66共5株菌分別染小鼠巨噬細(xì)胞RAW264.7,繪制其在細(xì)胞內(nèi)的生長曲線,比較變異菌株在巨噬細(xì)胞內(nèi)生存能力的變化。用酚水法和試劑盒法分別提取羊種布魯氏菌標(biāo)準(zhǔn)株16M的脂多糖,以商品化的脂多糖標(biāo)準(zhǔn)品(提取自EscherichiaColi)作為對照,對提取的脂多糖通過聚丙烯酰胺凝膠電泳及銀染比較其純度和結(jié)構(gòu)的異同。通過鱟試劑凝集試驗(yàn)檢測提取的脂多糖活性,并從操作過程、安全程度等方面比較兩種脂多糖提取方法的特點(diǎn)。用試劑盒法對4株布魯氏菌變異株提取脂多糖,并與光滑型和粗糙型布魯氏菌的脂多糖結(jié)構(gòu)進(jìn)行比較,通過聚丙烯酰胺凝膠電泳和銀染分析其結(jié)構(gòu)的差異。用IlluminaHiSeq對4株變異菌株進(jìn)行全基因組測序,對21個(gè)參與布魯氏菌脂多糖合成的基因(17個(gè)基因位于Ⅰ號染色體上,4個(gè)基因位于Ⅱ號染色體上)用BLAST軟件進(jìn)行序列比對,分析基因序列的改變及其引起的相應(yīng)基因產(chǎn)物的變化(以布魯氏菌標(biāo)準(zhǔn)株16M作為參考,其序列來自GenBank,結(jié)合各基因的功能分析其變化對脂多糖合成的可能影響。結(jié)果:1.在侵染后4小時(shí),16M和M5侵染組的IL-1β和IL-6的產(chǎn)生水平具有統(tǒng)計(jì)學(xué)意義。在各時(shí)間點(diǎn)上兩組的IL-12均無明顯變化。2.在各時(shí)間點(diǎn)上,巨噬細(xì)胞內(nèi)16M的數(shù)量均高于M5,且隨著時(shí)間推移二者的差距逐漸增加。3.四株變異布魯氏菌經(jīng)三勝黃素凝集試驗(yàn)和吖啶黃凝集試驗(yàn)發(fā)現(xiàn)其變異程度并不完全相同,035和033與兩種試劑均發(fā)生明顯的凝集反應(yīng),其凝集水平和粗糙犬種菌基本一致,而186和031兩株菌則僅與丫啶黃素發(fā)生較弱的凝集反應(yīng),和三勝黃素幾乎不發(fā)生凝集反應(yīng)。4.侵染后2小時(shí),粗糙型菌株033、035侵入數(shù)量高于光滑型菌株16M。031和186兩菌株侵入數(shù)量和]6M相近。5.侵染后48小時(shí),033、035、186三個(gè)菌株在細(xì)胞內(nèi)的數(shù)量和16M比較接近,而031菌株的數(shù)量和M5接近。6.酚水法和試劑盒法提取的LPS相比,在分子量為17kDa及26kDa處存在明顯的缺失。兩種方法提取的LPS中經(jīng)考馬斯亮藍(lán)染色均未顯示蛋白條帶,LPS純度較高。7.酚水法需要細(xì)菌量大,一般需要50g濕菌,試劑盒法需要菌量少,2 ml(菌懸液即可提取LPS。8.酚水法提取脂多糖必須提前對布魯氏菌滅活以保證安全,試劑盒法所需菌量少,且在生物安全柜內(nèi)操作,不需要提前滅活細(xì)菌。9.酚水法實(shí)驗(yàn)操作復(fù)雜,至少需要4天時(shí)間,試劑盒法操作簡單,需要一小時(shí)即可完成。10.酚水法提取的脂多糖純度較高,但會損失部分小分子量的LPS,試劑盒法提取的脂多糖更完整。11.兩種方法提取的布魯氏菌16M的LPS的鱟試劑凝集活性沒有明顯差別,均在5 EU/ng左右,而LPS標(biāo)準(zhǔn)品的凝集活性只有3.325 EU/ng。12.033和035兩菌株的脂多糖條帶更接近于RM6/66,條帶主要分布在17 kDa附近。13.186和031兩菌株的脂多糖在結(jié)構(gòu)上和光滑型布魯氏菌更相近,除在17kDa附近有條帶分布外,在34-43kDa,55-95kDa及180kDa這3個(gè)分子量附近也有明顯的條帶分布。14.在21個(gè)脂多糖合成相關(guān)基因中,有7個(gè)基因在四株變異菌株中菌未發(fā)生突變。wbk-E和manA兩個(gè)基因僅發(fā)生同義突變。大多數(shù)基因發(fā)生了一個(gè)或幾個(gè)核苷酸的錯義突變。四株變異布魯氏菌中存在較多的相同的點(diǎn)突變。15.有兩株變異布魯氏菌存在特有的基因突變:在033菌株檢測到wzt基因發(fā)生點(diǎn)突變,引起編碼氨基酸的改變,該基因參與編碼布魯氏菌ABC轉(zhuǎn)運(yùn)系統(tǒng),其變異影響O側(cè)鏈從細(xì)胞內(nèi)向外膜的轉(zhuǎn)運(yùn)。在035菌株,檢測到在manBcore基因第1334個(gè)核苷酸處插入了一段長度為27 bp的核苷酸序列引起移碼突變,該基因突變可能影響脂多糖核心寡糖成分的合成。結(jié)論:1.不同毒力布魯氏菌在巨噬細(xì)胞內(nèi)的存活能力具有明顯差別,可能作為體外檢測布魯氏菌毒力的方法,但需要更多的菌株進(jìn)行驗(yàn)證。2.16M和M5對巨噬細(xì)胞產(chǎn)生IL-1β、IL-6和IL-12的影響差別較小,雖然部分結(jié)果有統(tǒng)計(jì)學(xué)意義,但不能用作布魯氏菌毒力強(qiáng)弱的判斷指標(biāo)。3.試劑盒法提取布魯氏菌脂多糖更簡便、安全性高、成分更加完整。4.布魯氏菌光滑型向粗糙型的變異,提高了感染早期布魯氏菌侵入細(xì)胞的能力。5.布魯氏菌LPS合成相關(guān)基因變異性較大,不同的粗糙型變異株可能存在不同的變異機(jī)制。6.maBcore和wzt基因?qū)S持布魯氏菌LPS的完整具有重要作用,該基因突變可能會導(dǎo)致粗糙型變異。
[Abstract]:Objective: to establish a model of Brucella infection by macrophages, compare the invasion and intracellular viability of 4 strains of Brucella mutants to macrophages, and compare the characteristics and genetic changes of 4 strains of Brucella mutants. Methods: to establish a virulent strain of Brucella 16M and a weakly poisonous strain of Brucella vaccine. The strain M5 infected the mouse macrophage RAW264.7 cell model and used the PBS treatment group as the control. The expression level of IL-1 beta, IL-6 and IL-12 in the 4,8,24h cell supernatant after infection was measured by ELISA method, and the number of Brucella in the macrophages of 2,4,8,24 and 48h macrophages after infection was measured by the coated plate count, and the endogenous macrophage of Brucella was produced. The survival curves of Brucella strain 16M and vaccine strain M5 in macrophages were compared, and the survival curve model of Brucella in macrophages was established. The RAW264.7 of macrophages in mice was stained with 4 strains of mutated Brucella strain 2014186201403520150312015033 and 5 strains of Brucella canine, respectively. The growth curve of the mutant strain in the macrophage was compared. The lipopolysaccharide of the standard strain 16M of the sheep was extracted by the phenol water method and the kit method, and the commercialized lipopolysaccharide standard (extracted from EscherichiaColi) was used as the illumination, and the extracted lipopolysaccharide was passed through polyacrylamide gel. The similarities and differences of purity and structure were compared between electrophoresis and silver staining. The lipopolysaccharide activity was detected by limulus reagent agglutination test, and the characteristics of the extraction methods of two kinds of lipopolysaccharides were compared from operation process and safety. 4 strains of Brucella mutants were extracted with kits, and the fat of Brucella was more than that of smooth and rough Brucella. The structure of sugar structure was compared by polyacrylamide gel electrophoresis and silver staining. The whole genome of 4 mutant strains was sequenced with IlluminaHiSeq. 21 genes (17 genes on chromosome I and 4 genes on chromosome II) were sequenced by BLAST software. Comparison, analysis of the changes in the gene sequence and the changes in the corresponding gene products (with the standard strain 16M of Brucella as a reference, the sequence comes from GenBank, combining the functions of each gene to analyze the possible effects of its changes on the synthesis of lipopolysaccharide. Results: 1. at 4 hours after the infection, the production level of IL-1 beta and IL-6 in 16M and M5 infection groups has the same level. " Study significance. There was no obvious change in the IL-12 of the two groups at all time points. The number of 16M in the macrophages was higher than that of M5 at all time points, and the gap between the two and the.3. four strains of Brucella increased with the time of time, and the variation of Brucella was not exactly the same, 035 and 033, by the three vicintexin agglutination test and the acridine yellow coagulant set test. There were obvious agglutination reactions with the two reagents, and the agglutination level was basically the same as that of the crude canine species, while the 186 and 031 two strains had only a weaker agglutination reaction with the acridiaflavin, and the three vicine flavin almost did not have the agglutination reaction of.4. 2 hours after the infection, and the invasion number of the coarse strain 033035 was higher than the smooth strain 16M.031 and 186 two bacteria. The number of three strains and the number of three strains in the cell were close to 16M 48 hours after.5. invasion, and the number of 031 strains and the number of M5 were close to 17kDa and 26kDa at the molecular weight, compared with the.6. phenol water method and the LPS extracted by the reagent box method. The two methods extracted from the two methods were not stained with Coomassie blue. To show the protein band, the high purity of the LPS.7. water method requires a large amount of bacteria, usually needs 50g humid bacteria, the kit method needs less bacteria, 2 ml (the bacterial suspension can extract the LPS.8. phenol water method to extract LPS in advance to ensure the safety of Brucella inactivation. The reagent box method needs less bacteria, and operation in biological safety cabinet, do not need to be put out ahead of time. " The experimental operation of the active bacteria.9. phenol water method is complicated. It takes at least 4 days, the reagent box method is easy to operate, it takes one hour to complete the high purity of the lipopolysaccharide extracted by the.10. phenol water method, but it will lose the LPS of some small molecular weight, the LPS extracted by the kit method is more complete and the LPS of the 16M from the 16M of Brucella extracted by the two methods is agglutinated. There was no significant difference in activity, at about 5 EU/ng, while the agglutination activity of the LPS standard was only 3.325 EU/ng.12.033 and 035 two, the lipopolysaccharide bands were closer to the RM6/66. The bands of LPS mainly distributed near the 17 kDa and.13.186 and 031 two were more similar in structure to the smooth Brucella, except for the strip distribution near 17kDa. In the vicinity of the 3 molecular weights of 34-43kDa, 55-95kDa and 180kDa, there is a clear stripe distribution.14. in the 21 lipopolysaccharide synthesis related genes, and 7 genes have no mutation.Wbk-E and two genes in four mutant strains. Most of the genes have a missense mutation of one or several nucleotides. Four strain changes. There are many same point mutations in ISO brucellus.15., two strains of mutant Brucella have unique genetic mutation. The mutation of WZT gene is detected in 033 strains, causing the change of encoded amino acids. The gene is involved in encoding the ABC transport system of Brucella, and its variation affects the transfer of the O side chain from the inner membrane to the outer membrane. In 035 bacteria, the mutation affects the 035 bacteria. The strain, which was detected at 1334th nucleotides of the manBcore gene, inserted a sequence of nucleotide sequences with a length of 27 BP, which may affect the synthesis of the oligosaccharide composition of the lipopolysaccharide core. Conclusion: the survival ability of 1. different virulent brucellus in macrophages is significantly different, and may be used for detection of bru in vitro. Methods of virulence, but more strains are needed to verify that.2.16M and M5 have little difference in the effects of IL-1 beta, IL-6 and IL-12 on macrophages, although some results are statistically significant, but they can not be used as a criterion for the virulence of Brucella. The.3. kits are more convenient, safer and more ingredients. The whole.4. Brucella is smooth to rough type, which improves the ability of Brucella invaded early infection.5., the relative genes of Brucella LPS synthesis are larger. Different rough variant strains may have different mutation mechanisms,.6.maBcore and WZT genes are important for maintaining the integrity of Brucella LPS. Mutation may lead to rough variation.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.7

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