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  本文選題：伯氏疏螺旋體 + 基因克隆�。� 參考：《昆明醫(yī)科大學》2012年博士論文

【摘要】：研究目的： 1.用實時熒光定量PCR確定伯氏疏螺旋體感染小鼠模型不同組織的病原體載量,探討組織螺旋體載量與相應(yīng)組織病理損傷的關(guān)系。 2.建立伯氏疏螺旋體感染的小鼠模型,通過DECAL和微陣歹(microarray)等技術(shù)研究伯氏疏螺旋體在小鼠關(guān)節(jié)組織中的關(guān)節(jié)特異性基因表達譜,尋找伯氏螺旋體在關(guān)節(jié)中特異性高表達的基因。 3.以伯氏疏螺旋體標準株B31株基因組DNA為模板,PCR擴增在關(guān)節(jié)中特異性高表達的bmpA全基因序列,定向克隆和高效表達重組蛋白BmpA(rBmpA)并純化。對伯氏疏螺旋體BmpA蛋白進行亞細胞定位研究。 4.通過用重組伯氏疏螺旋體膜蛋白A (rBmpA)定期局部注射昆明(KM)小鼠脛跗關(guān)節(jié),誘導并建立萊姆關(guān)節(jié)炎(Lyme arthritis)昆明(KM)小鼠模型,在此基礎(chǔ)上,從形態(tài)學、影像學、病理學水平探索關(guān)節(jié)炎病變,檢測萊姆關(guān)節(jié)炎小鼠血清及關(guān)節(jié)液中Th-17細胞相關(guān)細胞因子IL-6、TGF-β及IL-17細胞因子含量,探討Th-17細胞在萊姆關(guān)節(jié)炎致病機制中作用。 5.從免疫細胞和分子水平初步研究rBmpA的致病機理。 研究內(nèi)容： 1.培養(yǎng)低傳代伯氏疏螺旋體至對數(shù)生長期,稀釋為1×105/ml。為建立伯氏疏螺旋體感染小鼠模型,于每只小鼠皮內(nèi)注射菌液100μl,在證實感染成功后,于第12d和18d分別取不同組織,提取總DNA,用實時熒光定量PCR分別測定組織中的螺旋體flaB基因拷貝數(shù),并標準化為每106β-肌動蛋白所對應(yīng)的flaB拷貝數(shù)(螺旋體數(shù))。對不同組織的螺旋體載量進行統(tǒng)計學處理,確定不同組織螺旋體載量是否有顯著性差異。 2.建立伯氏疏螺旋體感染小鼠模型,在不同時間點處死小鼠,收集小鼠關(guān)節(jié)、心臟、皮膚和膀胱組織,從四種組織中分別提取總RNA；隨后,用DECAL技術(shù)和微陣列技術(shù)分析不同時間點伯氏疏螺旋體在四種組織中的轉(zhuǎn)錄組；最后,將伯氏疏螺旋體在關(guān)節(jié)中的轉(zhuǎn)錄組依次與其他三種組織中的轉(zhuǎn)錄組進行比較,從而獲得伯氏疏螺旋體不同時間點的關(guān)節(jié)特異性基因表達譜,找出僅在關(guān)節(jié)中表達的伯氏疏螺旋體基因。 3.(1) BmpA基因的定向克隆與重組菌的產(chǎn)生：以伯氏疏螺旋體標準株B31株基因組DNA為模板,設(shè)計定向克隆引物,PCR擴增bmpA全基因序列,將bmpA基因定向克隆入表達載體pGEX-6p-1,酶切鑒定,轉(zhuǎn)化大腸桿菌BL21菌株,獲得bmpA重組菌。(2)重組bmpA高效表達與純化：從重組菌培養(yǎng)溫度,誘導時間,誘導劑的劑量,OD600等方面優(yōu)化誘導條件,找到高效表達重組bmpA的最佳方案。用GSH柱純化重組bmpA,探索純化bmpA的最佳條件。(3)bmpA的亞細胞定位研究。 4.(1)研究rBmpA在誘導萊姆關(guān)節(jié)炎致病中作用：優(yōu)化rBmpA注射濃度、劑量、次數(shù),探索rBmpA在誘導萊姆關(guān)節(jié)炎中的致病作用。(2)嘗試在新的動物品系(KM小鼠)、新方法(局部關(guān)節(jié)注射)建立萊姆關(guān)節(jié)炎動物模型的可能性：探索采取局部注射KM小鼠脛跗關(guān)節(jié),建立萊姆關(guān)節(jié)炎模型的方法。(3)從形態(tài)學、影像學、病理學水平探索關(guān)節(jié)炎病變。(4)探討Th-17細胞相關(guān)細胞因子IL-6、IL-17及TGF-β細胞因子在萊姆關(guān)節(jié)炎致病機理中作用：檢測萊姆關(guān)節(jié)炎小鼠血清及關(guān)節(jié)液中與Th-17細胞相關(guān)細胞因子IL-6、IL-17及TGF-β細胞因子含量,探討Th-17細胞在萊姆關(guān)節(jié)炎致病機制中作用。 5.研究rBmpA對小鼠腹腔巨噬細胞和脾臟淋巴細胞的活化作用。 研究結(jié)果： 1.在所檢測的4種代表性組織中,膀胱伯氏疏螺旋體載量在兩個典型時間點均最高,皮膚和關(guān)節(jié)次之,心臟最低。 2.與其他組織相比,在感染后第15天,伯氏疏螺旋體在小鼠關(guān)節(jié)組織特異地表達21個基因,其中13個基因位于伯氏疏螺旋體染色體上,8個基因位于質(zhì)粒上；在感染后的第105天,伯氏疏螺旋體在小鼠關(guān)節(jié)組織特異地表達24個基因,其中13個基因位于伯氏疏螺旋體染色體上,11個基因位于質(zhì)粒上。在關(guān)節(jié)中表達最高的是bmpA、bmpB等基因。 3. bmpA基因的克隆與表達研究。(1)在基因水平和蛋白水平上,都出現(xiàn)了目的條帶和目標峰,確定表達載體bmpA-pGEX-6p-1構(gòu)建成功,并表達重組bmpA。(2)通過分析比較,重組質(zhì)粒在37℃, IPTG誘導濃度為0.1mmol/ml,誘導時間為6小時,菌液的0D值為0.5-1.0以及在LB培養(yǎng)基上培養(yǎng)GST-bmpA融合蛋白表達量達到最大。(3)在最佳表達條件下1L的重組菌能純化到2.9-3.1mg的bmpA蛋白。(4)亞細胞定位研究表明,BmpA存在于螺旋體表面。 4. BmpA誘導關(guān)節(jié)炎研究。(1)用rBmpA稀釋液局部注射KM小鼠脛跗關(guān)節(jié),成功建立了萊姆關(guān)節(jié)炎(Lyme arthritis)-昆明(KM)小鼠模型,拓展了新的萊姆關(guān)節(jié)炎動物造模方法。(2)從形態(tài)學、影像學、病理學水平證實rBmpA與萊姆關(guān)節(jié)炎發(fā)病密切相關(guān)。(3)檢測萊姆關(guān)節(jié)炎小鼠血清及關(guān)節(jié)液中與Th-17細胞相關(guān)細胞因子IL-6、IL-17及TGF-β細胞因子含量與對照組及正常組比較無明顯統(tǒng)計學意義,分析和探討了相關(guān)原因,為未來進一步研究奠定了基礎(chǔ)。 5. rBmpA對小鼠腹腔巨噬細胞的作用不明顯,但對小鼠脾淋巴細胞有明顯的刺激增殖作用,并可以刺激脾淋巴細胞產(chǎn)生炎性細胞因子白細胞介素6。 結(jié)論： 1.伯氏疏螺旋體感染小鼠后,不同組織螺旋體載量有顯著性差異,膀胱螺旋體載量在兩個典型時間點均最高,皮膚和關(guān)節(jié)次之,心臟最低。組織螺旋體載量與組織損傷程度無密切關(guān)系。 2.伯氏疏螺旋體在小鼠關(guān)節(jié)組織中存在獨特的基因表達譜,其中螺旋體膜蛋白基因bmpA和bmpB在關(guān)節(jié)表達最高。這些在關(guān)節(jié)中特異表達的基因可能與萊姆關(guān)節(jié)炎的發(fā)生、發(fā)展有關(guān)。 3.(1)成功的構(gòu)建了表達重組蛋白bmpA的大腸桿菌原核表達系統(tǒng),并且在基因水平和蛋白水平上得到鑒定。(2)找到了高效表達重組BmpA的最佳方案。用GSH柱純化重組BmpA,探索出純化重組BmpA的最適條件。(3)亞細胞定位研究表明,BmpA存在于螺旋體細胞外膜表面。 4.(1)用rBmpA稀釋液局部注射KM小鼠脛跗關(guān)節(jié),成功建立了萊姆關(guān)節(jié)炎(Lyme arthritis)-昆明(KM)小鼠模型。(2)rBmpA與萊姆關(guān)節(jié)炎發(fā)病密切相關(guān)。 5. rBmpA對小鼠脾淋巴細胞有明顯的刺激增殖作用,并可以刺激脾淋巴細胞產(chǎn)生炎性細胞因子白細胞介素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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R377

【參考文獻】

中國期刊全文數(shù)據(jù)庫 前10條

1 譚毓繪;牛曉珊;卡力比努爾;于魯海;劉涌;孫荷;莫合塔爾;龍江;李紅燕;瑪衣努爾;李R，

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