羊种布鲁氏菌变异株细胞内存活能力及LPS遗传特征研究

发布时间：2018-05-16 01:48

本文选题：羊种布鲁氏菌 + 巨噬细胞　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：目的:建立布鲁氏菌侵染巨噬细胞模型,比较4株羊种布鲁氏菌变异株对巨噬细胞侵袭能力及胞内生存能力,比较4株羊种布鲁氏菌变异株的脂多糖特点及其遗传学变化。方法:建立强毒菌株羊种布鲁氏菌16M和弱毒菌株羊种布鲁氏菌疫苗菌株M5侵染小鼠巨噬细胞RAW264.7细胞模型,以PBS处理组作为对照,用ELISA法测量侵染后4、8、24h细胞上清液中IL-1β、IL-6和IL-12的表达水平;通过涂平板计数测量侵染后2、4、8、24和48h巨噬细胞内的布鲁氏菌的数量变化,绘制布鲁氏菌巨噬细胞内生存曲线,比较羊种布鲁氏菌强毒株16M和疫苗株M5在巨噬细胞内存活能力的差异,建立布鲁氏菌在巨噬细胞内的生存曲线模型。用4株羊种布鲁氏菌变异菌株2014186、2014035、2015031、2015033及犬种布鲁氏菌RM6/66共5株菌分别染小鼠巨噬细胞RAW264.7,绘制其在细胞内的生长曲线,比较变异菌株在巨噬细胞内生存能力的变化。用酚水法和试剂盒法分别提取羊种布鲁氏菌标准株16M的脂多糖,以商品化的脂多糖标准品(提取自EscherichiaColi)作为对照,对提取的脂多糖通过聚丙烯酰胺凝胶电泳及银染比较其纯度和结构的异同。通过鲎试剂凝集试验检测提取的脂多糖活性,并从操作过程、安全程度等方面比较两种脂多糖提取方法的特点。用试剂盒法对4株布鲁氏菌变异株提取脂多糖,并与光滑型和粗糙型布鲁氏菌的脂多糖结构进行比较,通过聚丙烯酰胺凝胶电泳和银染分析其结构的差异。用IlluminaHiSeq对4株变异菌株进行全基因组测序,对21个参与布鲁氏菌脂多糖合成的基因(17个基因位于Ⅰ号染色体上,4个基因位于Ⅱ号染色体上)用BLAST软件进行序列比对,分析基因序列的改变及其引起的相应基因产物的变化(以布鲁氏菌标准株16M作为参考,其序列来自GenBank,结合各基因的功能分析其变化对脂多糖合成的可能影响。结果:1.在侵染后4小时,16M和M5侵染组的IL-1β和IL-6的产生水平具有统计学意义。在各时间点上两组的IL-12均无明显变化。2.在各时间点上,巨噬细胞内16M的数量均高于M5,且随着时间推移二者的差距逐渐增加。3.四株变异布鲁氏菌经三胜黄素凝集试验和吖啶黄凝集试验发现其变异程度并不完全相同,035和033与两种试剂均发生明显的凝集反应,其凝集水平和粗糙犬种菌基本一致,而186和031两株菌则仅与丫啶黄素发生较弱的凝集反应,和三胜黄素几乎不发生凝集反应。4.侵染后2小时,粗糙型菌株033、035侵入数量高于光滑型菌株16M。031和186两菌株侵入数量和]6M相近。5.侵染后48小时,033、035、186三个菌株在细胞内的数量和16M比较接近,而031菌株的数量和M5接近。6.酚水法和试剂盒法提取的LPS相比,在分子量为17kDa及26kDa处存在明显的缺失。两种方法提取的LPS中经考马斯亮蓝染色均未显示蛋白条带,LPS纯度较高。7.酚水法需要细菌量大,一般需要50g湿菌,试剂盒法需要菌量少,2 ml(菌悬液即可提取LPS。8.酚水法提取脂多糖必须提前对布鲁氏菌灭活以保证安全,试剂盒法所需菌量少,且在生物安全柜内操作,不需要提前灭活细菌。9.酚水法实验操作复杂,至少需要4天时间,试剂盒法操作简单,需要一小时即可完成。10.酚水法提取的脂多糖纯度较高,但会损失部分小分子量的LPS,试剂盒法提取的脂多糖更完整。11.两种方法提取的布鲁氏菌16M的LPS的鲎试剂凝集活性没有明显差别,均在5 EU/ng左右,而LPS标准品的凝集活性只有3.325 EU/ng。12.033和035两菌株的脂多糖条带更接近于RM6/66,条带主要分布在17 kDa附近。13.186和031两菌株的脂多糖在结构上和光滑型布鲁氏菌更相近,除在17kDa附近有条带分布外,在34-43kDa,55-95kDa及180kDa这3个分子量附近也有明显的条带分布。14.在21个脂多糖合成相关基因中,有7个基因在四株变异菌株中菌未发生突变。wbk-E和manA两个基因仅发生同义突变。大多数基因发生了一个或几个核苷酸的错义突变。四株变异布鲁氏菌中存在较多的相同的点突变。15.有两株变异布鲁氏菌存在特有的基因突变:在033菌株检测到wzt基因发生点突变,引起编码氨基酸的改变,该基因参与编码布鲁氏菌ABC转运系统,其变异影响O侧链从细胞内向外膜的转运。在035菌株,检测到在manBcore基因第1334个核苷酸处插入了一段长度为27 bp的核苷酸序列引起移码突变,该基因突变可能影响脂多糖核心寡糖成分的合成。结论:1.不同毒力布鲁氏菌在巨噬细胞内的存活能力具有明显差别,可能作为体外检测布鲁氏菌毒力的方法,但需要更多的菌株进行验证。2.16M和M5对巨噬细胞产生IL-1β、IL-6和IL-12的影响差别较小,虽然部分结果有统计学意义,但不能用作布鲁氏菌毒力强弱的判断指标。3.试剂盒法提取布鲁氏菌脂多糖更简便、安全性高、成分更加完整。4.布鲁氏菌光滑型向粗糙型的变异,提高了感染早期布鲁氏菌侵入细胞的能力。5.布鲁氏菌LPS合成相关基因变异性较大,不同的粗糙型变异株可能存在不同的变异机制。6.maBcore和wzt基因对维持布鲁氏菌LPS的完整具有重要作用,该基因突变可能会导致粗糙型变异。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.7

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