本文選題：單核細胞增生李斯特菌 + ncRNArli87基因�。� 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：單核細胞增生李斯特菌(Listeria monocytogenes,LM)是一種兼性胞內(nèi)寄生的革蘭氏陽性菌。LM主要通過消化道引起動物和人的感染,是一種重要的人畜共患病原菌,在臨床主要引起人和動物的腦膜炎、敗血癥及流產(chǎn)等,對畜牧業(yè)和人類生命健康造成巨大的威脅,被WHO列為四大食源性致病菌之一。在LM感染宿主的過程中需要眾多的毒力因子參與,這些毒力因子的編碼基因主要集中在LM致病島1(LIPI-1)和致病島2(LIPI-2)2個區(qū)域中,其中LIPI-1含有6個基因,依次為prfA、plcA、hly、mpl、actA、plcB;LIP-2又稱為內(nèi)化素小島,主要編碼一些小分子的內(nèi)化素。近年來的研究發(fā)現(xiàn),LM非編碼ncRNA在調(diào)控其生長、基因表達、糖代謝、金屬離子轉(zhuǎn)運、生物膜形成、胞內(nèi)寄生及環(huán)境應(yīng)激過程中也扮演了重要的角色,其調(diào)控模式已經(jīng)成為LM調(diào)控網(wǎng)絡(luò)中的最重要作用方式之一。根據(jù)ncRNAs的調(diào)控機制,LM ncRNAs可以分為3種:順式作用RNA(Cis-acting RNAs,如核糖開關(guān))、反義RNA(Anti-Sense RNAs,asRNAs)和反式編碼的小RNA(Trans-encoded small RNAs,sRNAs)。rli87基因?qū)儆趕RNA,然而目前有關(guān)rli87基因的生物學(xué)功能尚不清楚。本研究應(yīng)用同源重組技術(shù)構(gòu)建rli87基因缺失株LM-?rli87,研究了ncRNA rli87缺失對LM生長特性的影響,探討了rli87在LM環(huán)境應(yīng)激能力和致病性的調(diào)控作用。主要研究內(nèi)容及結(jié)果如下:1、單核細胞增生李斯特菌Rli87基因缺失株的構(gòu)建、鑒定及其生長特性研究根據(jù)Gen Bank登錄的LMEGD-e基因組序列(登錄號:AL591824),用DNAMAN軟件進行同源性分析,選擇保守區(qū)域,用Primer 5.0軟件設(shè)計擴增上、下游同源臂引物。利用PCR技分別擴增rli87基因上、下游同源臂,然后運用SOE-PCR技術(shù)融合上、下游同源臂,獲得rli87基因缺失片段。將pMD19-T-△rli87和pKSV7質(zhì)粒進行雙酶切,將rli87基因缺失片段插入到穿梭載體PKSV7上,構(gòu)建重組穿梭質(zhì)粒pKSV7-△rli87。用電轉(zhuǎn)化的方法將pKSV7-△rli87電轉(zhuǎn)化到LM EGD-e中,對陽性轉(zhuǎn)化子在氯霉素與42℃條件下傳代培養(yǎng)10代,獲得具有氯霉素抗性的單交換株;然后在無氯霉素42℃條件下傳代培養(yǎng)15代,得到只能擴增出一條584bp的目的片段無氯霉素抗性的雙交換基因重組缺失株LM-△rli87。在無氯霉素壓力37℃條件下傳達培養(yǎng)20代,經(jīng)PCR檢測,結(jié)果證實LM-△rli87缺失株具有良好的遺傳穩(wěn)定性。在37℃條件下培養(yǎng)LM EGD-e和LM-△rli87菌株,兩者生長差異不顯著(p0.05),該結(jié)果證實rli87基因缺失對37℃生長特性沒有影響。2、Rli87基因缺失對單核細胞增生李斯特菌環(huán)境應(yīng)激的影響將LM EGD-e強毒株和構(gòu)建的LM-△rli87缺失株于相同條件下培養(yǎng)后,分別測試在不同溫度、pH值、高滲透壓及氧化環(huán)境壓力條件下應(yīng)急反應(yīng),并對應(yīng)激相關(guān)基因的表達量進行檢測。選取低溫30℃和高溫42℃,測定相同培養(yǎng)條件下不同時間點的OD600nm值,并繪制不同溫度條件下的生長曲線。結(jié)果在不用溫度條件下,LM-△rli87生長速度明顯高于LMEGD-e(p0.05)。在不同pH值生長條件下,當(dāng)pH=9時兩株菌生長差異顯著(P0.05);在2%H2O2氧化環(huán)境中出現(xiàn)生長停滯,活菌量隨著時間的變化呈下降趨勢,但LM-△rli87活菌量始終低于LM EGD-e(P0.05);在pH=9時,與LM EGD-e菌株相比,LM-△rli87缺失株rsbV、rsbW、hpt、clpP、ctsR 5個應(yīng)激相關(guān)基因的表達量均上升,表明在堿性環(huán)境中rli87對這5個應(yīng)激相關(guān)基因具有調(diào)節(jié)作用。3、Rli87基因缺失對單核細胞增生李斯特菌致病力的影響將LM EGD-e與構(gòu)建的rli87基因缺失株分別感染巨噬細胞RAW264.7和BALB/C小鼠,測其胞內(nèi)細菌計數(shù)、細胞粘附率的計算以及存活小鼠的統(tǒng)計,通過Real-time RT-PCR毒力基因表達量檢測的方法檢測五個毒力相關(guān)因子的表達量,并通過溶血試驗分析缺失株與野毒株的致病性差異。結(jié)果顯示,與LM EGD-e相比,LM-△rli87缺失株的粘附率和侵襲力均下降;肝、脾載菌量減少;缺失株的半數(shù)致死量較野毒株升高了103個數(shù)量級;毒力基因hly和PrfA的轉(zhuǎn)錄水平顯著下降。溶血試驗測定結(jié)果發(fā)現(xiàn),與強毒株LM EGD-e相比,LM-△rli87的溶血能力明顯降低。由于LM的溶血能力由LIPI-I上的hly基因編碼,結(jié)果提示rli87基因?qū)ly基因的表達具有調(diào)控作用。本研究通過SOE-PCR技術(shù)與同源重組的方法成功構(gòu)建了rli87基因缺失株,并研究了Rli87基因缺失對LM環(huán)境應(yīng)激及致病力的影響。與LM EGD-e相比,Rli87基因缺失株的環(huán)境應(yīng)激能力下降,致病性減弱,由此表明,rli87基因?qū)毦玖哂幸欢ǖ恼{(diào)控作用,為揭示rli87調(diào)控LM毒力的分子機制奠定了前期基礎(chǔ)。
[Abstract]:Mononuclear cell proliferation List Rand (Listeria monocytogenes, LM) is a facultative intracellular parasitic gram positive bacteria,.LM, which mainly causes animal and human infection through the digestive tract. It is an important zoonosis, which mainly causes meningitis, septicaemia and abortion in humans and animals, and is healthy for animal husbandry and human life. WHO is one of the four major food borne pathogenic bacteria. In the process of LM infection, many virulence factors are needed. The coding genes of these virulence factors are mainly concentrated in the 2 regions of LM pathogenicity island 1 (LIPI-1) and pathogenetic Island 2 (LIPI-2), and LIPI-1 contains 6 genes, which are prfA, plcA, hly, MPL, actA, plcB; P-2, also known as the endogenous hormone Isle, mainly encodes some small molecules of the endogenous hormone. Recent studies have found that LM non coded ncRNA has also played an important role in regulating its growth, gene expression, glucose metabolism, metal ion transport, biofilm formation, intracellular parasitism and environmental stress, and its regulatory model has become a LM regulatory network. One of the most important ways of action is that, according to the regulatory mechanism of ncRNAs, LM ncRNAs can be divided into 3 kinds: the CIS action RNA (Cis-acting RNAs, such as ribose switch), the antisense RNA (Anti-Sense RNAs, asRNAs) and the trans encoding small RNA (Trans-encoded), but the biological function of the gene is not yet clear. Chu. In this study, the rli87 gene deletion strain LM-? Rli87 was constructed by homologous recombination technology. The effect of ncRNA rli87 deletion on the growth characteristics of LM was studied, and the regulation effect of rli87 on the environmental stress and pathogenicity of LM environment was discussed. The main contents and results are as follows: 1, the construction, identification and identification of the Rli87 gene deletion strain of Lester monocytic bacteria. The growth characteristics are based on the LMEGD-e genome sequence of Gen Bank (login number: AL591824), using DNAMAN software to analyze the homology, select the conservative region and amplify the downstream homologous arm primers with Primer 5 software. The PCR technique is used to amplify the rli87 gene and the lower reaches of the homologous arm, and then the downstream homology is fused with SOE-PCR technology. The rli87 gene deletion fragment was obtained. The pMD19-T- Delta rli87 and pKSV7 plasmid were double enzyme cut and the rli87 gene deletion fragment was inserted into the shuttle carrier PKSV7. The recombinant shuttle plasmid pKSV7- Delta rli87. was transformed into LM EGD-e by electrical transformation, and the positive transformants were cultured at chloramphenicol and 42 C under the condition of chloramphenicol and 42. In the 10 generation, a single exchange strain with chloramphenicol resistance was obtained, and then cultured for 15 generations under the condition of chloramphenicol at 42 centigrade, the two exchange gene LM- Delta rli87., which could only amplify a 584bp target fragment without chloramphenicol resistance, was transmitted for 20 generations under the condition of chloramphenicol pressure 37, and the result was confirmed by PCR, and the results confirmed that LM- delta RLI The 87 deletions had good genetic stability. The growth of LM EGD-e and LM- Delta rli87 strains at 37 C was not significantly different (P0.05). The results showed that the rli87 gene deletion had no effect on the growth characteristics of 37 degrees C, and the effect of the deletion of Rli87 gene on the environmental stress of monocytic Lester bacteria was a strong strain and construction of LM EGD-e. The LM- Delta rli87 deletion strain was cultured under the same condition and tested at different temperatures, pH, hypertonic pressure and oxidative stress, and detected the expression of stress related genes. The low temperature 30 and 42 C were selected to determine the OD600nm value at different time points under the same culture conditions, and the different temperature strips were drawn. Under the conditions of no temperature, the growth rate of LM- Delta rli87 was significantly higher than that of LMEGD-e (P0.05). Under the different pH growth conditions, the growth difference between two strains of bacteria was significant (P0.05), and the growth stagnated in the 2%H2O2 oxidation environment and the amount of living bacteria decreased with the change of time, but the LM- Delta rli87 living bacteria was always low. At LM EGD-e (P0.05), at pH=9, compared with LM EGD-e strain, LM- Delta rli87 missing strains rsbV, rsbW, HPT, and clpP, the expression of 5 stress related genes all increased, indicating that the 5 stress related genes were regulated in the alkaline environment. RAW264.7 and BALB/C mice were infected with the constructed rli87 gene, respectively. The count of intracellular bacteria, the calculation of cell adhesion rate and the statistics of the surviving mice were measured. The expression of five virulence related factors was detected by the detection of Real-time RT-PCR virulence gene expression, and the missing strains were analyzed by hemolysis test. The results showed that the adhesion and invasiveness of the LM- Delta rli87 missing strain were all decreased compared with LM EGD-e, the liver, the spleen carrying capacity decreased, the half lethal dose of the missing strain increased 103 orders of magnitude higher than the wild strain, and the transcription level of the virulence gene hly and PrfA decreased significantly. The results of hemolysis test found that it was found with the strong strain LM EGD. Compared with -e, the hemolysis ability of LM- Delta rli87 was significantly reduced. As the hemolysis ability of LM was encoded by the hly gene on LIPI-I, the results suggested that the rli87 gene had a regulatory effect on the expression of hly gene. This study successfully constructed the rli87 gene deletion strain by the method of SOE-PCR and homologous recombination, and studied the stress of Rli87 gene deletion on LM environment stress. And the effect of pathogenicity. Compared with LM EGD-e, the environmental stress ability of the Rli87 gene deletion strain decreased and the pathogenicity weakened. Thus, the rli87 gene had a certain regulating effect on the bacterial virulence, which laid a preliminary foundation for revealing the molecular mechanism of rli87 to regulate the toxicity of LM.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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