Rli87基因缺失对LM环境应激及致病力的影响

发布时间：2018-05-25 18:55

本文选题：单核细胞增生李斯特菌 + ncRNArli87基因　；参考：《石河子大学》2015年硕士论文

【摘要】：单核细胞增生李斯特菌(Listeria monocytogenes,LM)是一种兼性胞内寄生的革兰氏阳性菌。LM主要通过消化道引起动物和人的感染,是一种重要的人畜共患病原菌,在临床主要引起人和动物的脑膜炎、败血症及流产等,对畜牧业和人类生命健康造成巨大的威胁,被WHO列为四大食源性致病菌之一。在LM感染宿主的过程中需要众多的毒力因子参与,这些毒力因子的编码基因主要集中在LM致病岛1(LIPI-1)和致病岛2(LIPI-2)2个区域中,其中LIPI-1含有6个基因,依次为prfA、plcA、hly、mpl、actA、plcB;LIP-2又称为内化素小岛,主要编码一些小分子的内化素。近年来的研究发现,LM非编码ncRNA在调控其生长、基因表达、糖代谢、金属离子转运、生物膜形成、胞内寄生及环境应激过程中也扮演了重要的角色,其调控模式已经成为LM调控网络中的最重要作用方式之一。根据ncRNAs的调控机制,LM ncRNAs可以分为3种:顺式作用RNA(Cis-acting RNAs,如核糖开关)、反义RNA(Anti-Sense RNAs,asRNAs)和反式编码的小RNA(Trans-encoded small RNAs,sRNAs)。rli87基因属于sRNA,然而目前有关rli87基因的生物学功能尚不清楚。本研究应用同源重组技术构建rli87基因缺失株LM-?rli87,研究了ncRNA rli87缺失对LM生长特性的影响,探讨了rli87在LM环境应激能力和致病性的调控作用。主要研究内容及结果如下:1、单核细胞增生李斯特菌Rli87基因缺失株的构建、鉴定及其生长特性研究根据Gen Bank登录的LMEGD-e基因组序列(登录号:AL591824),用DNAMAN软件进行同源性分析,选择保守区域,用Primer 5.0软件设计扩增上、下游同源臂引物。利用PCR技分别扩增rli87基因上、下游同源臂,然后运用SOE-PCR技术融合上、下游同源臂,获得rli87基因缺失片段。将pMD19-T-△rli87和pKSV7质粒进行双酶切,将rli87基因缺失片段插入到穿梭载体PKSV7上,构建重组穿梭质粒pKSV7-△rli87。用电转化的方法将pKSV7-△rli87电转化到LM EGD-e中,对阳性转化子在氯霉素与42℃条件下传代培养10代,获得具有氯霉素抗性的单交换株;然后在无氯霉素42℃条件下传代培养15代,得到只能扩增出一条584bp的目的片段无氯霉素抗性的双交换基因重组缺失株LM-△rli87。在无氯霉素压力37℃条件下传达培养20代,经PCR检测,结果证实LM-△rli87缺失株具有良好的遗传稳定性。在37℃条件下培养LM EGD-e和LM-△rli87菌株,两者生长差异不显著(p0.05),该结果证实rli87基因缺失对37℃生长特性没有影响。2、Rli87基因缺失对单核细胞增生李斯特菌环境应激的影响将LM EGD-e强毒株和构建的LM-△rli87缺失株于相同条件下培养后,分别测试在不同温度、pH值、高渗透压及氧化环境压力条件下应急反应,并对应激相关基因的表达量进行检测。选取低温30℃和高温42℃,测定相同培养条件下不同时间点的OD600nm值,并绘制不同温度条件下的生长曲线。结果在不用温度条件下,LM-△rli87生长速度明显高于LMEGD-e(p0.05)。在不同pH值生长条件下,当pH=9时两株菌生长差异显著(P0.05);在2%H2O2氧化环境中出现生长停滞,活菌量随着时间的变化呈下降趋势,但LM-△rli87活菌量始终低于LM EGD-e(P0.05);在pH=9时,与LM EGD-e菌株相比,LM-△rli87缺失株rsbV、rsbW、hpt、clpP、ctsR 5个应激相关基因的表达量均上升,表明在碱性环境中rli87对这5个应激相关基因具有调节作用。3、Rli87基因缺失对单核细胞增生李斯特菌致病力的影响将LM EGD-e与构建的rli87基因缺失株分别感染巨噬细胞RAW264.7和BALB/C小鼠,测其胞内细菌计数、细胞粘附率的计算以及存活小鼠的统计,通过Real-time RT-PCR毒力基因表达量检测的方法检测五个毒力相关因子的表达量,并通过溶血试验分析缺失株与野毒株的致病性差异。结果显示,与LM EGD-e相比,LM-△rli87缺失株的粘附率和侵袭力均下降;肝、脾载菌量减少;缺失株的半数致死量较野毒株升高了103个数量级;毒力基因hly和PrfA的转录水平显著下降。溶血试验测定结果发现,与强毒株LM EGD-e相比,LM-△rli87的溶血能力明显降低。由于LM的溶血能力由LIPI-I上的hly基因编码,结果提示rli87基因对hly基因的表达具有调控作用。本研究通过SOE-PCR技术与同源重组的方法成功构建了rli87基因缺失株,并研究了Rli87基因缺失对LM环境应激及致病力的影响。与LM EGD-e相比,Rli87基因缺失株的环境应激能力下降,致病性减弱,由此表明,rli87基因对细菌毒力具有一定的调控作用,为揭示rli87调控LM毒力的分子机制奠定了前期基础。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【相似文献】