devR基因突變對結(jié)核分枝桿菌細胞毒性的影響
發(fā)布時間：2019-03-14 20:15
【摘要】：結(jié)核病的病死率僅次于HIV的感染,已經(jīng)嚴(yán)重威脅到了全人類的生存和健康。我國作為結(jié)核病負擔(dān)大國,結(jié)核分枝桿菌的耐藥問題也日漸突出,因此研究結(jié)核分枝桿菌的耐藥和基因突變機制,對于疾病的防控意義重大。課題組前期研究發(fā)現(xiàn)結(jié)核分枝桿菌雙組份系統(tǒng)的dev R(反應(yīng)調(diào)節(jié)子編碼基因)在臨床耐藥菌株中出現(xiàn)不同程度的升高表達現(xiàn)象,并利用同源重組方法構(gòu)建了dev R基因敲除的卡介苗菌株。本課題在前期工作基礎(chǔ)上,構(gòu)建了乙酰胺啟動子誘導(dǎo)表達dev R的回補菌株,并通過驗證dev R對巨噬細胞和上皮細胞的毒性作用,進一步探索了dev R缺失導(dǎo)致的休眠障礙與結(jié)核分枝桿菌細胞毒性的關(guān)系。一、dev R基因回補菌株構(gòu)建及鑒定1.構(gòu)建由乙酰胺啟動子誘導(dǎo)表達dev R的重組BCG菌株利用PJV53質(zhì)粒和Pcherry 3質(zhì)粒及目的基因,構(gòu)建乙酰胺啟動子啟動表達dev R的重組載體,電轉(zhuǎn)化入dev R敲除的BCG菌株感受態(tài)中,經(jīng)PCR、雙酶切和測序鑒定正確后,命名此重組菌株為dev R回補菌株(Pcherry3-Acetamindase-dev R)。2.重組BCG菌株P(guān)cherry3-Acetamindase-dev R生長特性的觀察將BCG野生菌株、dev R敲除菌株和dev R回補菌株分別培養(yǎng)至對數(shù)生長期即OD600=0.6時,調(diào)整菌量至105 CFU/m L,以菌液和培養(yǎng)基比例為1:50接種至含有各自抗生素抗性的7H9液體培養(yǎng)基中培養(yǎng),分別在第1-14 d測定OD600值,繪制細菌生長曲線。結(jié)果顯示,dev R敲除菌株在早期(2~10 d)生長較BCG野生菌株明顯加快(P0.01);dev R回補菌株和BCG野生菌株之間的生長無顯著性差異(P0.05)。3.三種菌株對異煙肼和利福平的耐藥發(fā)生率檢測將BCG野生菌株、dev R敲除菌株和dev R回補菌株以1個Mc Farland濃度接種到分別含有10倍MIC濃度的異煙肼和利福平的7H10固體培養(yǎng)基上培養(yǎng)28天,計數(shù)培養(yǎng)基上生長出的菌落數(shù),得到耐藥發(fā)生率。結(jié)果顯示,三種菌株對異煙肼和利福平的耐藥發(fā)生率均無顯著性差異(P0.05)。二、三種菌株分別感染上皮細胞A549和巨噬細胞Raw264.7的細胞毒性檢測三種菌株均以感染復(fù)數(shù)(MOI)等于10∶1分別感染上皮細胞A549巨噬細胞Raw264.7,分別采用流式細胞儀和酶聯(lián)免疫吸附試驗于0、24、48、72 h測定細胞凋亡率和乳酸脫氫酶(LDH)釋放水平;采用RT-PCR方法測定凋亡相關(guān)基因bcl-2和bad的表達。細胞感染試驗結(jié)果顯示,dev R敲除菌株感染A549細胞和Raw264.7細胞后,細胞的凋亡和壞死效應(yīng)明顯增加,相較于BCG野生菌株和dev R回補菌株差異顯著(P0.05);三種菌株感染A549細胞均導(dǎo)致凋亡相關(guān)基因bcl-2表達升高,并且dev R敲除菌株感染組明顯高于BCG和dev R回補菌株感染組(P0.05);三種菌株感染Raw264.7細胞均導(dǎo)致凋亡相關(guān)基因bad表達升高,并且dev R敲除菌株感染組明顯高于BCG野生菌株和dev R回補菌株感染組(P0.05)。本研究初步證明了dev R敲除的BCG菌株毒力明顯增強。
[Abstract]:The mortality rate of tuberculosis, second only to HIV infection, has seriously threatened the survival and health of all mankind. As a major burden of tuberculosis in China, the problem of drug resistance of Mycobacterium tuberculosis is becoming more and more prominent. Therefore, it is of great significance to study the mechanism of drug resistance and gene mutation of Mycobacterium tuberculosis in order to prevent and control the disease. Previous studies in our team found that the dev R (response regulator gene of Mycobacterium tuberculosis bicomponent system was expressed in different degrees in clinical drug-resistant strains, and the expression level of the two-component system of Mycobacterium tuberculosis was significantly higher than that of the control group. The Bacillus Calmette-Guerin (BCG) strain with dev R gene knockout was constructed by homologous recombination. On the basis of previous work, a complement strain of dev-R induced by acetamide promoter was constructed, and the toxicity of dev-R on macrophages and epithelial cells was verified. The relationship between dormancy disorder caused by dev R deletion and cytotoxicity of Mycobacterium tuberculosis was further investigated. Construction and Identification of a, dev R Gene complementing strain The recombinant BCG strain expressing dev R induced by acetamide promoter was constructed by using PJV53 plasmid, Pcherry 3 plasmid and target gene to construct the recombinant vector of dev R expression initiated by acetamide promoter. The recombinant vector was transformed into the competent state of dev R knockout strain BCG by electroporation, and the recombinant vector was transformed into the competent state of dev R knockout strain by PCR,. After being identified by double enzyme digestion and sequencing, the recombinant strain was named as dev R complementary strain (Pcherry3-Acetamindase-dev R). 2.) Observation on the growth characteristics of recombinant BCG strain Pcherry3-Acetamindase-dev R the wild BCG strain, dev R knockout strain and dev R supplementary strain were cultured to the logarithmic growth period (OD600=0.6) respectively, and the bacterial quantity was adjusted to 105 CFU/m / L. The bacteria were cultured in 7H9 liquid medium containing antibiotic resistance at the ratio of 1:50 to 1:50. OD 600 was measured on the 1st-14th day, and the growth curve of the bacteria was drawn. The results showed that the growth of, dev R knockout strain was significantly faster than that of BCG wild strain (P0.01) (P0.01). There was no significant difference in the growth of); dev R knockout strain and BCG wild strain (P0.05). Detection of resistance rates of three strains to isoniazid and rifampicin in wild strains of BCG The dev R knockout strain and the dev R complement strain were inoculated on 7H10 solid medium containing 10 times MIC concentration of isoniazid and rifampicin respectively in a Mc Farland concentration for 28 days. The number of colonies grown on the medium was counted and the incidence of drug resistance was obtained. The results showed that there was no significant difference in the rates of resistance to isoniazid and rifampicin among the three strains (P0.05). Cytotoxicity of three strains infected with A549 and Raw264.7 respectively; all of the three strains infected A549 cells with multiple numbers of (MOI) equal to 10:1, respectively, infected with Raw264.7, in epithelial cells A549 and macrophages, respectively. Cell apoptosis rate and lactate dehydrogenase (LDH) release were measured by flow cytometry and enzyme-linked immunosorbent assay (Elisa) at 0,24,48,72 h, respectively. The expression of apoptosis-related genes bcl-2 and bad was determined by RT-PCR. The results of cell infection test showed that the apoptosis and necrosis of A549 cells and Raw264.7 cells infected by, dev R knockout strain increased significantly, compared with wild strain BCG and dev R strain (P0.05). The expression of apoptosis-related gene bcl-2 was increased in A549 cells infected by three kinds of strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG and dev R backfill group (P0.05). The expression of apoptosis-related gene bad was increased in Raw264.7 cells infected by three strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG wild strain and dev R backfill strain group (P0.05). The results showed that the virulence of dev R knockout strain BCG was significantly enhanced.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5
本文編號：2440333
[Abstract]:The mortality rate of tuberculosis, second only to HIV infection, has seriously threatened the survival and health of all mankind. As a major burden of tuberculosis in China, the problem of drug resistance of Mycobacterium tuberculosis is becoming more and more prominent. Therefore, it is of great significance to study the mechanism of drug resistance and gene mutation of Mycobacterium tuberculosis in order to prevent and control the disease. Previous studies in our team found that the dev R (response regulator gene of Mycobacterium tuberculosis bicomponent system was expressed in different degrees in clinical drug-resistant strains, and the expression level of the two-component system of Mycobacterium tuberculosis was significantly higher than that of the control group. The Bacillus Calmette-Guerin (BCG) strain with dev R gene knockout was constructed by homologous recombination. On the basis of previous work, a complement strain of dev-R induced by acetamide promoter was constructed, and the toxicity of dev-R on macrophages and epithelial cells was verified. The relationship between dormancy disorder caused by dev R deletion and cytotoxicity of Mycobacterium tuberculosis was further investigated. Construction and Identification of a, dev R Gene complementing strain The recombinant BCG strain expressing dev R induced by acetamide promoter was constructed by using PJV53 plasmid, Pcherry 3 plasmid and target gene to construct the recombinant vector of dev R expression initiated by acetamide promoter. The recombinant vector was transformed into the competent state of dev R knockout strain BCG by electroporation, and the recombinant vector was transformed into the competent state of dev R knockout strain by PCR,. After being identified by double enzyme digestion and sequencing, the recombinant strain was named as dev R complementary strain (Pcherry3-Acetamindase-dev R). 2.) Observation on the growth characteristics of recombinant BCG strain Pcherry3-Acetamindase-dev R the wild BCG strain, dev R knockout strain and dev R supplementary strain were cultured to the logarithmic growth period (OD600=0.6) respectively, and the bacterial quantity was adjusted to 105 CFU/m / L. The bacteria were cultured in 7H9 liquid medium containing antibiotic resistance at the ratio of 1:50 to 1:50. OD 600 was measured on the 1st-14th day, and the growth curve of the bacteria was drawn. The results showed that the growth of, dev R knockout strain was significantly faster than that of BCG wild strain (P0.01) (P0.01). There was no significant difference in the growth of); dev R knockout strain and BCG wild strain (P0.05). Detection of resistance rates of three strains to isoniazid and rifampicin in wild strains of BCG The dev R knockout strain and the dev R complement strain were inoculated on 7H10 solid medium containing 10 times MIC concentration of isoniazid and rifampicin respectively in a Mc Farland concentration for 28 days. The number of colonies grown on the medium was counted and the incidence of drug resistance was obtained. The results showed that there was no significant difference in the rates of resistance to isoniazid and rifampicin among the three strains (P0.05). Cytotoxicity of three strains infected with A549 and Raw264.7 respectively; all of the three strains infected A549 cells with multiple numbers of (MOI) equal to 10:1, respectively, infected with Raw264.7, in epithelial cells A549 and macrophages, respectively. Cell apoptosis rate and lactate dehydrogenase (LDH) release were measured by flow cytometry and enzyme-linked immunosorbent assay (Elisa) at 0,24,48,72 h, respectively. The expression of apoptosis-related genes bcl-2 and bad was determined by RT-PCR. The results of cell infection test showed that the apoptosis and necrosis of A549 cells and Raw264.7 cells infected by, dev R knockout strain increased significantly, compared with wild strain BCG and dev R strain (P0.05). The expression of apoptosis-related gene bcl-2 was increased in A549 cells infected by three kinds of strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG and dev R backfill group (P0.05). The expression of apoptosis-related gene bad was increased in Raw264.7 cells infected by three strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG wild strain and dev R backfill strain group (P0.05). The results showed that the virulence of dev R knockout strain BCG was significantly enhanced.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5
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相關(guān)碩士學(xué)位論文 前1條
1 旦正尖措;devR基因突變對結(jié)核分枝桿菌細胞毒性的影響[D];第四軍醫(yī)大學(xué);2015年
，本文編號：2440333
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