伯氏疏螺旋体的莱姆关节炎相关毒力因子研究

发布时间：2018-06-18 22:12

  本文选题：伯氏疏螺旋体 + 基因克隆　； 参考：《昆明医科大学》2012年博士论文

【摘要】：研究目的： 1.用实时荧光定量PCR确定伯氏疏螺旋体感染小鼠模型不同组织的病原体载量,探讨组织螺旋体载量与相应组织病理损伤的关系。 2.建立伯氏疏螺旋体感染的小鼠模型,通过DECAL和微阵歹(microarray)等技术研究伯氏疏螺旋体在小鼠关节组织中的关节特异性基因表达谱,寻找伯氏螺旋体在关节中特异性高表达的基因。 3.以伯氏疏螺旋体标准株B31株基因组DNA为模板,PCR扩增在关节中特异性高表达的bmpA全基因序列,定向克隆和高效表达重组蛋白BmpA(rBmpA)并纯化。对伯氏疏螺旋体BmpA蛋白进行亚细胞定位研究。 4.通过用重组伯氏疏螺旋体膜蛋白A (rBmpA)定期局部注射昆明(KM)小鼠胫跗关节,诱导并建立莱姆关节炎(Lyme arthritis)昆明(KM)小鼠模型,在此基础上,从形态学、影像学、病理学水平探索关节炎病变,检测莱姆关节炎小鼠血清及关节液中Th-17细胞相关细胞因子IL-6、TGF-β及IL-17细胞因子含量,探讨Th-17细胞在莱姆关节炎致病机制中作用。 5.从免疫细胞和分子水平初步研究rBmpA的致病机理。 研究内容： 1.培养低传代伯氏疏螺旋体至对数生长期,稀释为1×105/ml。为建立伯氏疏螺旋体感染小鼠模型,于每只小鼠皮内注射菌液100μl,在证实感染成功后,于第12d和18d分别取不同组织,提取总DNA,用实时荧光定量PCR分别测定组织中的螺旋体flaB基因拷贝数,并标准化为每106β-肌动蛋白所对应的flaB拷贝数(螺旋体数)。对不同组织的螺旋体载量进行统计学处理,确定不同组织螺旋体载量是否有显著性差异。 2.建立伯氏疏螺旋体感染小鼠模型,在不同时间点处死小鼠,收集小鼠关节、心脏、皮肤和膀胱组织,从四种组织中分别提取总RNA；随后,用DECAL技术和微阵列技术分析不同时间点伯氏疏螺旋体在四种组织中的转录组；最后,将伯氏疏螺旋体在关节中的转录组依次与其他三种组织中的转录组进行比较,从而获得伯氏疏螺旋体不同时间点的关节特异性基因表达谱,找出仅在关节中表达的伯氏疏螺旋体基因。 3.(1) BmpA基因的定向克隆与重组菌的产生：以伯氏疏螺旋体标准株B31株基因组DNA为模板,设计定向克隆引物,PCR扩增bmpA全基因序列,将bmpA基因定向克隆入表达载体pGEX-6p-1,酶切鉴定,转化大肠杆菌BL21菌株,获得bmpA重组菌。(2)重组bmpA高效表达与纯化：从重组菌培养温度,诱导时间,诱导剂的剂量,OD600等方面优化诱导条件,找到高效表达重组bmpA的最佳方案。用GSH柱纯化重组bmpA,探索纯化bmpA的最佳条件。(3)bmpA的亚细胞定位研究。 4.(1)研究rBmpA在诱导莱姆关节炎致病中作用：优化rBmpA注射浓度、剂量、次数,探索rBmpA在诱导莱姆关节炎中的致病作用。(2)尝试在新的动物品系(KM小鼠)、新方法(局部关节注射)建立莱姆关节炎动物模型的可能性：探索采取局部注射KM小鼠胫跗关节,建立莱姆关节炎模型的方法。(3)从形态学、影像学、病理学水平探索关节炎病变。(4)探讨Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子在莱姆关节炎致病机理中作用：检测莱姆关节炎小鼠血清及关节液中与Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子含量,探讨Th-17细胞在莱姆关节炎致病机制中作用。 5.研究rBmpA对小鼠腹腔巨噬细胞和脾脏淋巴细胞的活化作用。 研究结果： 1.在所检测的4种代表性组织中,膀胱伯氏疏螺旋体载量在两个典型时间点均最高,皮肤和关节次之,心脏最低。 2.与其他组织相比,在感染后第15天,伯氏疏螺旋体在小鼠关节组织特异地表达21个基因,其中13个基因位于伯氏疏螺旋体染色体上,8个基因位于质粒上；在感染后的第105天,伯氏疏螺旋体在小鼠关节组织特异地表达24个基因,其中13个基因位于伯氏疏螺旋体染色体上,11个基因位于质粒上。在关节中表达最高的是bmpA、bmpB等基因。 3. bmpA基因的克隆与表达研究。(1)在基因水平和蛋白水平上,都出现了目的条带和目标峰,确定表达载体bmpA-pGEX-6p-1构建成功,并表达重组bmpA。(2)通过分析比较,重组质粒在37℃, IPTG诱导浓度为0.1mmol/ml,诱导时间为6小时,菌液的0D值为0.5-1.0以及在LB培养基上培养GST-bmpA融合蛋白表达量达到最大。(3)在最佳表达条件下1L的重组菌能纯化到2.9-3.1mg的bmpA蛋白。(4)亚细胞定位研究表明,BmpA存在于螺旋体表面。 4. BmpA诱导关节炎研究。(1)用rBmpA稀释液局部注射KM小鼠胫跗关节,成功建立了莱姆关节炎(Lyme arthritis)-昆明(KM)小鼠模型,拓展了新的莱姆关节炎动物造模方法。(2)从形态学、影像学、病理学水平证实rBmpA与莱姆关节炎发病密切相关。(3)检测莱姆关节炎小鼠血清及关节液中与Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子含量与对照组及正常组比较无明显统计学意义,分析和探讨了相关原因,为未来进一步研究奠定了基础。 5. rBmpA对小鼠腹腔巨噬细胞的作用不明显,但对小鼠脾淋巴细胞有明显的刺激增殖作用,并可以刺激脾淋巴细胞产生炎性细胞因子白细胞介素6。 结论： 1.伯氏疏螺旋体感染小鼠后,不同组织螺旋体载量有显著性差异,膀胱螺旋体载量在两个典型时间点均最高,皮肤和关节次之,心脏最低。组织螺旋体载量与组织损伤程度无密切关系。 2.伯氏疏螺旋体在小鼠关节组织中存在独特的基因表达谱,其中螺旋体膜蛋白基因bmpA和bmpB在关节表达最高。这些在关节中特异表达的基因可能与莱姆关节炎的发生、发展有关。 3.(1)成功的构建了表达重组蛋白bmpA的大肠杆菌原核表达系统,并且在基因水平和蛋白水平上得到鉴定。(2)找到了高效表达重组BmpA的最佳方案。用GSH柱纯化重组BmpA,探索出纯化重组BmpA的最适条件。(3)亚细胞定位研究表明,BmpA存在于螺旋体细胞外膜表面。 4.(1)用rBmpA稀释液局部注射KM小鼠胫跗关节,成功建立了莱姆关节炎(Lyme arthritis)-昆明(KM)小鼠模型。(2)rBmpA与莱姆关节炎发病密切相关。 5. rBmpA对小鼠脾淋巴细胞有明显的刺激增殖作用,并可以刺激脾淋巴细胞产生炎性细胞因子白细胞介素6。
[Abstract]:The purpose of the study is:
1. the amount of pathogens in different tissues of burgospira burgdorferi infected mice was determined by real time fluorescence quantitative PCR, and the relationship between the load of tissue helix and the corresponding histopathological damage was investigated.
2. to establish a mouse model of Borrelia burgdorferi infection, the specific gene expression profiles of burgospira burgdorferi in the joint tissues of mice were studied by DECAL and microarray (microarray), and the specific high expression genes of burgospira burgdorferi were found in the joint.
3. the genomic DNA of the standard strain B31 strain of burgospira burgdorferi was used as a template, and PCR was amplified in the specific high expression of bmpA gene sequence in the joint, directed clones and highly expressed recombinant protein BmpA (rBmpA) and purified. The subcellular localization of bergospira BmpA protein was studied.
4. by regular local injection of the tibial and tarsal joints of Kunming (KM) mice with recombinant bursiostomy membrane protein A (rBmpA), the induced and established Kunming (KM) mice model of Lyme arthritis (Lyme arthritis) was induced. On this basis, the pathological changes were explored from morphology, imaging and pathological level, and Th-17 in the serum and joint fluid of Lyme arthritis mice was detected by Th-17. Cell related cytokines IL-6, TGF- beta and IL-17 cytokine levels, and explore the role of Th-17 cells in the pathogenesis of Lyme arthritis.
5. preliminary study on pathogenesis of rBmpA from immune cell and molecular level.
Research content:
1. to develop a low biography of Treponema sparsely to a logarithmic growth period and 1 x 105/ml. to establish a mouse model of Borrelia burgdorferi infection, and 100 mu L in each mouse's intradermal injection. After confirming the infection, the different tissues were taken from 12D and 18D, respectively, and the total DNA was extracted, and the real-time quantitative PCR was used to determine the flaB base of the spiral body in the tissue. As a result of the copy number, the number of flaB copies (the number of helix) corresponding to every 106 beta actin was standardized. The load of helix in different tissues was statistically treated to determine whether there was a significant difference in the load of different tissue helix.
2. a mouse model of Borrelia burgdorferi was established, mice were killed at different time points, mouse joints, heart, skin and bladder tissues were collected, total RNA was extracted from four tissues. Then, DECAL technique and microarray technique were used to analyze the transcriptional groups of burgospira burgdorferi at different time points in four tissues; finally, bersine thinning spiral was used. The transcriptional groups in the joints were compared with those in the other three tissues in order to obtain the specific gene expression profiles of burgospira burgdorferi at different time points, and to find out the bersate Borrelia gene expressed only in the joints.
3. (1) BmpA gene directed cloning and the production of recombinant bacteria: Based on the genomic DNA of the standard strain B31 strain of Borrelia burgdorferi, design directed clone primers, PCR amplification of bmpA whole gene sequence, cloning bmpA gene into expression vector pGEX-6p-1, enzymatic identification, transformation of Bacillus large Enterobacter BL21 strain and obtaining bmpA recombinant bacteria. (2) high efficiency of recombinant bmpA Expression and purification: optimize the induction conditions from the culture temperature of the recombinant bacteria, the induction time, the dose of the inducer, OD600 and so on, and find the best way to express the recombinant bmpA efficiently. The optimum conditions for the purification of the recombinant bmpA with the GSH column are used to explore the optimum conditions for the purification of bmpA. (3) the subcellular localization of bmpA.
4. (1) study the role of rBmpA in inducing Lyme arthritis: optimize the rBmpA injection concentration, dose, and number of times, explore the pathogenicity of rBmpA in the induction of Lyme arthritis. (2) try to establish a new animal strain (KM mouse), a new method (local joint injection) to establish a leam arthritis animal model: explore the local injection of KM small The method of establishing the rat tibial and tarsal joint to establish the Lyme arthritis model. (3) to explore the disease of arthritis from morphological, imaging and pathological levels. (4) to explore the role of Th-17 cell related cytokines IL-6, IL-17 and TGF- beta cytokine in the pathogenesis of Lyme arthritis: the detection of serum and joint fluid in Lyme arthritis mice is related to Th-17 cells Cytokine IL-6, IL-17 and TGF- beta cytokines were used to investigate the role of Th-17 cells in the pathogenesis of Lyme arthritis.
5. to study the activation of rBmpA on mouse peritoneal macrophages and splenic lymphocytes.
The results of the study:
1. in the 4 representative tissues examined, the Burr's Borrelia burgdorferi load was the highest in two typical time points, followed by the skin and joint, and the heart was the lowest.
2. compared with other tissues, fifteenth days after infection, 21 genes were expressed in the joint tissues of the mouse's joint tissue, of which 13 genes were located on the Borrelia burgdorferi chromosome and 8 genes were located on the plasmid. In the 105th day after infection, burgospira burgdorferi expressed 24 genes in the joint tissues of the mice, 13 of them. On the chromosome of Borrelia burgdorferi, 11 genes are located on the plasmid. The highest expression in the joint is bmpA, bmpB and other genes.
The cloning and expression of 3. bmpA gene. (1) at the gene level and protein level, the target band and target peak were found, and the expression vector bmpA-pGEX-6p-1 was successfully constructed, and the recombinant bmpA. (2) was expressed by analysis and comparison. The recombinant plasmid was at 37, the induced concentration of IPTG was 0.1mmol/ml, the induction time was 6 hours, and the 0D value of the bacterial liquid was 0.5-1.0 And the maximum expression of GST-bmpA fusion protein was reached on the LB medium. (3) the recombinant bacteria of 1L could be purified to the bmpA protein of 2.9-3.1mg under the optimum conditions. (4) the subcellular localization study showed that BmpA existed on the surface of the helix.
4. BmpA induced arthritis study. (1) the KM mouse tibialis tarsies were injected locally with rBmpA diluents, and Lyme arthritis (Lyme arthritis) Kunming (KM) mouse model was successfully established. (2) the morphological, imaging, and pathological level confirmed that rBmpA was closely related to the incidence of Lyme arthritis. (3) detection The content of cytokines IL-6, IL-17 and TGF- beta cytokine related to Th-17 cells in the serum and joint fluid of the mice with Lyme arthritis had no significant statistical significance compared with the control group and the normal group. The related reasons were analyzed and discussed, which laid the foundation for further research.
The effect of 5. rBmpA on mouse peritoneal macrophages is not obvious, but it can stimulate the proliferation of spleen lymphocytes and stimulate the inflammatory cytokines interleukin 6. in spleen lymphocytes.
Conclusion:
After 1. Borrelia infected mice, the load of helix in different tissues was significantly different. The load of the spiral body of the bladder was the highest at two typical time points, and the skin and joint were the lowest and the heart was the lowest. The load of tissue helix was not closely related to the degree of tissue injury.
2. Borrelia burgdorferi has a unique gene expression profile in mouse joint tissues, in which the highest expression of spiral membrane protein gene bmpA and bmpB. These genes specifically expressed in the joint may be related to the occurrence and development of Lyme arthritis.
3. (1) successfully constructed the Escherichia coli prokaryotic expression system expressing the recombinant protein bmpA and identified the gene level and protein level. (2) the best way to express recombinant BmpA was found. The optimum conditions for the purification of recombinant BmpA with GSH column were used to explore the optimum conditions for the purification of the recombinant BmpA. (3) the subcellular localization study showed that BmpA existed in the spiral. The surface of the outer membrane of the somatic cell.
4. (1) local injection of KM tibial and tarsal joints with rBmpA diluents, a mouse model of Lyme arthritis (Lyme arthritis) Kunming (KM) was successfully established. (2) rBmpA is closely related to the incidence of Lyme arthritis.
5. rBmpA can stimulate the proliferation of spleen lymphocytes in mice and stimulate the production of inflammatory cytokines interleukin 6. in splenic lymphocytes.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R377

【参考文献】

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