本文選題：傷寒沙門菌 + mig-14��； 參考：《江蘇大學》2017年碩士論文

【摘要】：目的:mig-14是沙門菌屬通過水平轉移獲得的外來毒力基因,其產物蛋白Mig-14在沙門菌屬拮抗多粘菌素B殺傷的過程中扮演著重要的角色。并且作為一種調節(jié)因子,Mig-14可調控多種基因表達。但是,對于mig-14的表達機制與特性仍然未明。本研究旨在探索不同環(huán)境中傷寒沙門菌中mig-14基因的表達特性。方法:1.傷寒沙門菌缺失變異株的制備使用自殺質粒法,利用同源重組的原理,分別制備傷寒沙門菌調節(jié)因子Hil D、Him A、Him D、Ydg T、Hha、HNS的缺失變異株,Fis及Omp R缺失變異株由實驗室保存。2.q RT-PCR分析mig-14轉錄水平采用實時定量PCR技術,觀察傷寒沙門菌野生株及各缺陷變異株中mig-14基因的轉錄水平。普通LB(Luria-Bertani培養(yǎng)基)培養(yǎng)條件下,比較傷寒沙門菌野生株及ydg T、hha、hns缺失變異株中mig-14基因的轉錄水平,從而判斷mig-14作為傷寒沙門菌水平轉移獲得的外來基因,其轉錄是否被沉默蛋白(Ydg T、Hha、HNS)抑制;多粘菌素B(polymyxin,PB)刺激條件下,比較傷寒沙門菌野生株及hil D、him A、him D、fis、omp R缺失變異株中mig-14的轉錄水平,從而判斷mig-14在PB存在的情況下,其轉錄是否由調節(jié)因子Hild、Him A、Him D、Fis、Omp R激活。3.Western blot免疫印跡法檢測Mig-14表達水平實驗室存有Mig-14兔多克隆抗體,收集傷寒沙門菌及各缺失變異株全菌蛋白,以Mig-14兔多克隆抗體進行Western blot免疫印跡實驗從而分析Mig-14在各菌株中的表達情況。4.lacZ融合實驗構建mig-14啟動子區(qū)域的lacZ基因融合質粒pHRP309。PCR擴增mig-14啟動子區(qū)域,與含β-半乳糖苷酶基因(lac Z)的質粒載體p HRP309連接構建mig-14啟動子-lac Z融合質粒。將融合質粒導入傷寒沙門菌野生株和各缺失變異株中,細菌胞內的β-半乳糖苷酶活性即可表示mig-14的轉錄表達水平。裂解細菌,收集上清,測定β-半乳糖苷酶活性,以Miller Units表示。5.蛋白表達及純化利用大腸桿菌BL21系統(tǒng),分別表達純化沉默蛋白HNS以及宿主整合因子IHF的α亞基Him A,以進一步分析調節(jié)因子對mig-14的調控作用。6.凝膠阻滯實驗(EMSA)通過體外凝膠阻滯實驗(EMSA)分析調節(jié)因子HNS、Him A對mig-14表達的調節(jié)機制。7.分析IHF、Mig-14對iagA的影響構建傷寒沙門菌的himA/mig-14雙基因缺失變異株,構建him A和mig-14的p BAD33異源表達載體,雙缺失變異株分別回補him A和mig-14后,觀察高滲早期iag A基因的轉錄情況。結果:1.傷寒沙門菌基因缺失變異株的制備成功制備傷寒沙門菌hil D、him A、him D、hha及ydg T的缺失變異株,并經序列鑒定分析驗證;傷寒沙門菌hns的缺失變異株構建失敗,遂通過構建傷寒沙門菌hns高表達株反向分析HNS對mig-14的抑制。2.普通LB培養(yǎng)條件下沉默蛋白抑制mig-14轉錄利用實時定量PCR(q RT-PCR)比較傷寒沙門菌野生株及hha、ydg T缺失變異株及hns高表達株中的mig-14的轉錄水平。結果顯示hha、ydg T缺失變異株中mig-14轉錄水平明顯高于傷寒沙門菌野生株,且缺失了hha后mig-14轉錄水平上升明顯高于ydg T缺失變異株。另外,傷寒沙門菌GIFU10007的hns基因無法敲除,故通過構建hns高表達菌株來反映HNS對mig-14的抑制作用。結果顯示,傷寒沙門菌hns高表達菌株的mig-14轉錄水平明顯低于野生株。這表明,沉默蛋白HNS、Hha、Ydg T抑制外來基因mig-14的轉錄表達。3.PB刺激下野生株及各缺失變異株中mig-14轉錄水平PB可刺激mig-14表達。實時定量PCR及l(fā)ac Z融合實驗結果均顯示:在PB刺激下,傷寒沙門菌的hil D、him A、him D、fis、omp R缺失變異株同野生株相比,mig-14轉錄表達水平均明顯下降。結果表明,在PB刺激下,傷寒沙門菌調節(jié)因子Hil D、Fis、IHF(Him A/Him D)、Omp R均參與激活mig-14。4.Western blot免疫印跡法檢測Mig-14表達水平利用實驗室前期工作制備的Mig-14兔多克隆抗體,檢測各菌株中Mig-14表達水平。結果發(fā)現傷寒沙門菌mig-14缺失變異株及野生株在目的條帶處(Mig-14大小約36.5 KD)無明顯差異條帶,故認為Mig-14兔多克隆抗體失效。5.成功表達傷寒沙門菌HNS蛋白及IHF的α亞基Him A成功構建p ET28a-HNS、p ET15b-Him A及p ET15b-Him D表達載體,利用大腸桿菌BL21表達系統(tǒng),成功表達并純化得到HNS及Him A蛋白(Him D形成包涵體,嘗試多次后均無法得到有效活性形式)。6.凝膠阻滯實驗(EMSA)結果顯示,HNS及Him A都能與mig-14基因的啟動子區(qū)域結合,形成DNA-蛋白復合物,延緩DNA遷移速率。這也意味著,調節(jié)因子HNS及Him A能直接作用于mig-14的啟動子區(qū)域發(fā)揮調節(jié)作用。7.高滲早期宿主整合因子(IHF)激活mig-14表達可增加iagA的轉錄成功構建傷寒沙門菌him A/mig-14雙缺失變異株。分別回補him A和mig-14后發(fā)現,在高滲早期,回補him A后,iag A轉錄水平同雙缺陷相比明顯上升;而回補mig-14后,iag A轉錄水平同雙缺陷相比并無明顯統(tǒng)計學差異。結果表明,高滲早期,IHF激活mig-14與iag A,Mig-14也能激活iag A,但Mig-14發(fā)揮激活iag A的作用依賴IHF的存在。結論:mig-14作為傷寒沙門菌水平轉移獲得的外來基因,其表達受沉默蛋白HNS、Hha、Ydg T抑制;多粘菌素B刺激下,調節(jié)因子Hil D、Fis、IHF(Him A/Him D)、Omp R均參與激活mig-14的轉錄表達。沉默蛋白HNS及宿主整合因子(IHF)的α亞基Him A能直接與mig-14的啟動子結合,調節(jié)mig-14轉錄。本實驗室前期工作發(fā)現Mig-14在高滲早期大量表達并能影響SPI-1上基因的表達,本次工作發(fā)現Mig-14在高滲早期對iag A及下游致病島1的調節(jié)作用依賴于宿主整合因子(IHF)。這些證據初步揭示了Mig-14作為一種新的調節(jié)因子在復雜的細菌調節(jié)網絡中的角色。
[Abstract]:Objective: mig-14 is a foreign virulence gene obtained by the level transfer of Salmonella, and its product protein Mig-14 plays an important role in the process of inhibiting the killing of polymyxin B by Salmonella. And as an regulatory factor, Mig-14 can regulate the expression of multiple genes. However, the mechanism and characteristics of mig-14 are still unknown. The aim of this study is to explore the expression characteristics of mig-14 gene in Salmonella typhi in different environments. Methods: the preparation of the deletion mutant of 1. Salmonella typhi was prepared by using the suicide plasmid method. Using the principle of homologous recombination, Hil D, Him A, Him D, Ydg T, Hha, HNS mutant strain were prepared respectively. The transcriptional level of the mig-14 gene in the wild Salmonella typhi strain and the defective variants was observed by real-time quantitative PCR technique, and the transcriptional level of the mig-14 gene in the wild Salmonella typhi and the defective variants was observed by.2.q RT-PCR analysis. The transcriptional level of the mig-14 genes in the wild strains of Salmonella typhi and YDG T, HHA, HNS deletion mutants were compared under the normal LB (Luria-Bertani medium) culture. To judge whether the transcription of mig-14 as an alien gene of Salmonella typhi was suppressed by Ydg T (Hha, HNS), and the transcriptional level of the wild strains of Salmonella typhi and HIL D and him A were compared under the conditions of polymyxin (PB). In the case, whether the transcriptional factor Hild, Him A, Him D, Fis, Omp R activated.3.Western blot to detect the Mig-14 rabbit polyclonal antibody in the Mig-14 expression laboratory, and collect the total bacterial protein of Salmonella typhimurium and the missing mutant strain. The expression of -14 in each strain.4.lacZ fusion experiment constructed the lacZ gene fusion plasmid pHRP309.PCR of the mig-14 promoter region to amplify the mig-14 promoter region and construct the mig-14 promoter -lac fusion plasmid with the plasmid carrier P HRP309 containing the beta galactosidase gene (LAC Z). The fusion plasmid was introduced into the wild Salmonella typhi strain and the wild strain. Among the missing variants, the activity of beta galactosidase in bacterial cell could indicate the transcriptional expression level of mig-14. Lysate bacteria, collect supernatant, determine the activity of beta galactosidase, express the expression of.5. protein by Miller Units and use the BL21 system of Escherichia coli to express the purified silencing protein HNS and the host integrator IHF respectively. Subunit Him A, in order to further analyze the regulatory role of regulatory factors on mig-14,.6. gel block experiment (EMSA) through in vitro gel block test (EMSA) analysis of regulatory factor HNS, Him A's regulation mechanism for mig-14 expression.7. analysis IHF. 14 P BAD33 heterologous expression vector, double deletion mutants were added to him A and mig-14 to observe the transcriptional situation of IAG A gene in early hypertonic. Results: the preparation of the gene deletion mutant of Salmonella typhi was successfully prepared to prepare HIL D, him A, him him A, and the sequence identification and validation; Salmonella typhi. The failure of the deletion mutant of HNS was constructed by constructing the HNS high expression strain of Salmonella typhimurium in reverse analysis of the inhibition of mig-14 by HNS and the inhibition of mig-14 transcriptional utilization of mig-14 transcriptional using real-time quantitative PCR (Q RT-PCR) compared with the wild strains of Salmonella typhi and HHA, YDG deletion mutant and high expression strain of the transcriptional water The results showed that the mig-14 transcriptional level in the HHA YDG T deletion mutant was significantly higher than that of the wild Salmonella typhi strain, and the mig-14 transcriptional level increased significantly higher than that of the YDG T deletion mutant. In addition, the HNS gene of the GIFU10007 Salmonella typhi could not be knocked out, so the inhibitory effect of HNS to HNS was reflected by the construction of the HNS high expression strain. The results showed that the mig-14 transcriptional level of the HNS high expression strain of Salmonella typhi was significantly lower than that of the wild strain. This indicated that the silencing protein HNS, Hha, Ydg T inhibited the transcription of the foreign gene mig-14, and the mig-14 transcriptional level PB stimulated mig-14 expression in the wild strain and the missing variant strains. Under the stimulation of PB, the HIL D, him A, him D, FIS, OMP R deletion mutants of the Salmonella typhi were significantly lower than those of the wild strains. The Mig-14 rabbit polyclonal antibody prepared by the early laboratory work was used to detect the Mig-14 expression level in each strain. The results showed that the mig-14 deletion mutant and the wild strain of Salmonella typhi had no distinct difference bands at the destination strip (Mig-14 size about 36.5 KD). Therefore, the Mig-14 rabbit polyclonal antibody failed.5. to successfully express the HNS of Salmonella typhimurium. The protein and the alpha subunit Him A of IHF successfully construct P ET28a-HNS, P ET15b-Him A and P ET15b-Him D expression vector, and use the Escherichia coli BL21 expression system. Combining with the promoter region of the mig-14 gene to form a DNA- protein complex and delay the migration rate of DNA, it also means that the regulatory factor HNS and Him A can directly act on the promoter region of mig-14 and play a regulatory role in the early stage of.7. hyperosmotic host integration factor (IHF) activation mig-14, which can increase iagA transcriptional success for the construction of Salmonella typhi him A/mig-14 double deletion mutants. After supplementing him A and mig-14, the transcriptional level of IAG A increased obviously in the early stage of hypertonic, after the recharge of him A, while the IAG A transcription level was not significantly different from those of the double defects. But the role of Mig-14 to activate IAG A depends on the existence of IHF. Conclusion: mig-14 is a foreign gene obtained by the horizontal transfer of Salmonella typhi, its expression is inhibited by the silent protein HNS, Hha, Ydg T; under the stimulation of the polymyxin B, the regulatory factor Hil D is involved in the activation of the transcriptional expression. The alpha subunit Him A of the combination factor (IHF) can directly bind to the promoter of mig-14 and regulate mig-14 transcription. Earlier work in our laboratory found that Mig-14 was expressed in early hypertonic and could affect the expression of genes on SPI-1. This work found that the regulation of Mig-14 on IAG A and downstream pathogeny island 1 in early hypertonic is dependent on host integration factor (IHF). These evidence preliminarily reveals the role of Mig-14 as a new regulatory factor in complex bacterial regulatory networks.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378

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