基于srtA的Lm细胞壁差异蛋白的筛选及lmo2714基因缺失株的构建

发布时间：2018-04-26 09:22

本文选题：Lm + 蛋白组学　；参考：《石河子大学》2017年硕士论文

【摘要】：单核细胞增多性李斯特菌(listeria monocytogenes,Lm)是能够穿越人和动物三大屏障从而引起人和动物发病的一种食源性致病菌,主要引起败血症、胃肠炎、脑膜炎等临床症状,Lm的发病率不高,但死亡率较高。在Lm细胞胞壁上存在许多与细菌毒力密切相关的表面蛋白,其中有一类蛋白,羧基端带有LPXTG基序,并且被分选酶A(Sortase A,Srt A)识别,通过共价的方式锚定到肽聚糖上,呈现在细菌表面。本研究以临床分离株LM90SB2(绵羊脑组织中单核细胞增多性李斯特菌,4b血清型)为研究对象,利用蛋白组学技术对LM90SB2和Srt A缺失株的细胞壁蛋白进行差异蛋白的筛选,运用生物信息学分析差异蛋白可能的功能;对细胞壁表面蛋白Lmo2714构建缺失株,研究其在细胞粘附与侵袭,小鼠毒力试验,环境适应性等生物学特性中的作用,为挖掘新的细胞壁表面蛋白以及致病机制奠定基础。1.变溶菌素(Mutanolysin)酶解Lm细胞壁最佳条件的筛选:为了提高酶解法结合TCA-丙酮沉淀法提取临床分离株LB90SB2细胞壁表面蛋白的效率,本试验对变溶菌素的浓度及酶解时间进行了优化。选择20μg/mL,60μg/mL的变溶菌素浓度进行30min-4 h,37℃酶解条件及37℃和4℃低温联合酶解的条件,通过测定酶解前后OD600、CFU的变化,以此判断细胞膜的完整性和计算原生质体形成率,筛选出最佳酶解浓度及酶解时间的条件。在酶浓度的最佳条件下,再测定不同酶解时间段细胞壁蛋白的浓度再测定不同酶解时间段细胞壁蛋白提取浓度。结果表明,在酶解30 min-4 h时间段内,原生质体形成率可以从0提高到91.00%,细胞壁蛋白浓度从0.272 mg/mL增加到1.735mg/mL。△OD600在1.86%-14.50%变动,但△OD600的统计学处理差异不显著。变溶菌素浓度在60μg/m L时,作用4 h,原生质体的形成率能够达到91.00%,远远高于20μg/mL的变溶菌素同样作用4 h的78%。最终,60μg/mL为变溶菌素浓度,4h的酶解时间,是酶解LM90SB2细胞壁的最佳条件。这为下一步研究细胞壁蛋白筛选奠定重要基础。2.LM90SB2与LM90SB2 srtA基因缺失株(LM90SB2-△srtA)细胞壁差异蛋白筛选:为进一步探明由srtA分选的细胞壁表面蛋白谱,本试验采用非标记定量技术(Lable-free)结合液相色谱-串联质谱(LC-MS/MS)技术筛选差异蛋白,通过生物信息学分析,对差异蛋白进行功能预测。结果显示,利用非标记定量技术结合LC-MS/MS,共检测出蛋白组数697个,肽段数3367个,差异显著蛋白36个,其中20个蛋白在亲本株中检测到,在缺失株未检出;7个蛋白在缺失株检测到的,在亲本株未检出;7个蛋白在亲本株中的蛋白量大于缺失株(P0.05),2个蛋白在亲本株中的蛋白量小于缺失株(P0.05);生物信息学分析,这36个差异显著蛋白与内化素、细胞壁锚定蛋白等有关。srtA分选蛋白谱的探明,为挖掘新的毒力因子或药物靶标奠定了基础。3.LM90SB2-△lmo2714缺失株的构建:为成功构建LM90SB2-△lmo2714,本试验利用同源重组技术。首先设计lmo2714基因上下臂特异性引物,通过PCR技术扩增,接着通过SOE-PCR技术得到lmo2714基因的缺失片段即△lmo2714,然后与pMD19-T连接,将测序正确的△lmo2714再与pKSV7连接,pKSV7-△lmo2714电转化LM90SB2感受态细胞中,阳性转化子在高温和氯霉素抗性的双重压力条件下连续传代,使之发生同源重组,同时用旁外侧引物(lmo2714 dele-1和lmo2714 dele-2)PCR检测缺失片段,最后置于无抗性的30℃条件下获得无质粒的LM90SB2-△lmo2714,连续传代后检测缺失株稳定性。结果显示:利用旁侧引物检测构建的缺失株,可以观察到大小为1033bp的条带,而亲本株的条带大小为1981bp,盲传20代之后,没有发生返祖现象。表明成功构建LM90SB2-△lmo2714缺失株且遗传稳定性表现良好。4.LM90SB2-△lmo2714缺失株的生物学特性:为探究Lmo2714的功能,将亲本株作为对照,检测菌株的生物膜形成能力、生化特性、对环境的适应性、对小鼠的致病性及对SIEC,MBMEC,RAW264.7和HBMEC的粘附、侵袭能力及胞内增殖情况。结果显示:缺失株对环境的适应性以及生物膜形成能力没有明显差异;影响部分生化特性;但LM90SB2-△lmo2714小鼠LD50比亲本株高1.34个对数级(P0.05),感染小鼠72h后的肝、脾、脑脏器中的含菌量极显著低于亲本株(P0.01);显著降低了对MBMEC和RAW264.7细胞的侵袭能力(P0.05)以及对MBMEC的粘附能力(P0.05);提高了对SIEC的粘附能力(P0.05);但是胞内增殖差异不显著。推测Lmo2714与Lm的致病性有一定关系,能部分影响Lm的毒力。
[Abstract]:Listeria monocytogenes (Lm) is a food borne pathogenic bacteria that can cause human and animal disease to pass through the three major barrier of human and animal, which mainly causes the clinical symptoms of septicemia, gastroenteritis, meningitis and so on. The incidence of Lm is not high, but the mortality rate is high. There are many and bacterial venom on the cell wall of Lm cell. A closely related surface protein, with a class of protein, carboxyl terminus with LPXTG motif, and identified by the sorting enzyme A (Sortase A, Srt A), anchored to peptidoglycan and on the bacterial surface by covalently. This study was studied by clinical isolates of LM90SB2 (monocytic monocytic Lester, 4b serotype in sheep brain group). Elephants, using proteomics technology to screen the cell wall proteins of LM90SB2 and Srt A deletion strains, use bioinformatics to analyze the possible functions of differential proteins; construct missing strains on cell wall surface protein Lmo2714, and study the biological properties of cell adhesion and invasion, mouse toxicity test, environmental adaptability and so on. In order to screen the best conditions for the.1. lysozyme (Mutanolysin) Lm cell wall, in order to excavate the new cell wall surface protein and the pathogenic mechanism, in order to improve the efficiency of extracting the surface protein of the LB90SB2 cell wall of clinical isolates with TCA- acetone precipitation method, the concentration of mutant lysozyme and the time of enzymolysis were carried out in this experiment. Optimization. The concentration of 20 g/mL and 60 mu g/mL was selected for 30min-4 h, 37 C and 37 and 4 centigrade at low temperature. By measuring the changes of OD600 and CFU before and after the enzymolysis, the integrity of the cell membrane and the formation rate of protoplast were calculated, and the optimum enzyme concentration and the enzyme hydrolysis time were screened out. Under the optimum conditions, the concentration of cell wall protein in different enzymatic time segments was measured and the cell wall protein extraction concentration in different enzymatic time segments was measured. The results showed that the protoplast formation rate could be increased from 0 to 91% in the 30 min-4 h time period, and the cell wall protein concentration increased from 0.272 mg/mL to 1.735mg/mL. Delta OD600 in 1.86%-14.5. 0% changes, but there is no significant difference in the statistical treatment of delta OD600. When the concentration of lysozyme is 60 g/m L, the effect is 4 h, the formation rate of protoplast can reach 91%, which is far higher than that of 20 mu g/mL, which also acts as the 4 h 78%., and 60 Mu g/mL is the concentration of lysozyme and 4H's enzymolysis time, which is the best condition for the enzyme hydrolysis of LM90SB2 cell wall. In order to study cell wall protein screening for the next step,.2.LM90SB2 and LM90SB2 srtA gene deletion strain (LM90SB2- Delta srtA) cell wall differential protein screening: to further identify the protein spectrum of cell wall surface by srtA, using non labeling quantitative technique (Lable-free) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) technology The difference protein was predicted by bioinformatics analysis. The results showed that the number of protein groups was 697, the number of peptide segments was 697, the number of peptide segments was 3367, and the difference was 36, of which 20 proteins were detected in the parent strain, and the missing strains were not detected; 7 proteins were detected in the missing strains. The number of protein in the parent strain was larger than that of the missing strain (P0.05), and the protein amount of the 2 protein in the parent strain was less than the missing strain (P0.05). Bioinformatics analysis, the 36 differences of the protein and the protein of the cell wall anchoring protein and other.SrtA proteins were explored to dig new virulence factors. Or the drug target laid the construction of the basic.3.LM90SB2- Delta lmo2714 deletion strain: to construct LM90SB2- Delta lmo2714 successfully, this experiment uses the homologous recombination technology. First, the specific primers of the lmo2714 gene are designed and amplified by PCR technology. Then the deletion fragment of the lmo2714 gene is obtained by SOE-PCR technology, which is then Delta lmo2714, and then pMD19-T with pMD19-T. Connection, the right Delta lmo2714 was sequenced and then connected with pKSV7, and pKSV7- Delta lmo2714 was electrically converted to LM90SB2 receptive cells. The positive transformants were continuously subcultured under the double pressure condition of high temperature and chloramphenicol resistance, and the homologous recombination was made, and the missing fragments were detected by the lateral primers (lmo2714 dele-1 and lmo2714 dele-2) PCR, and finally placed at the same time. LM90SB2- Delta lmo2714 without plasmid was obtained at 30 degrees centigrade without resistance, and the stability of the missing strains was detected after continuous passage. The results showed that the size of 1033bp was observed by using side primers and the size of the parent strain was 1981bp. After the blind transmission of the 20 generation, no reversion was found. It showed that LM was successfully constructed. The 90SB2- Delta lmo2714 deletion strain and the genetic stability showed good biological characteristics of.4.LM90SB2- Delta lmo2714 deletion strain: To explore the function of Lmo2714, the parent strain was used as the control to detect the biofilm formation ability, biochemical characteristics, environmental adaptability, the pathogenesis of mice and the adhesion to SIEC, MBMEC, RAW264.7 and HBMEC. The results showed that there was no obvious difference in the adaptability of the environment and the ability of biofilm formation in the missing strains, and some biochemical characteristics were affected, but the LD50 of LM90SB2- Delta lmo2714 mice was 1.34 higher than that of the parent strain (P0.05), and the bacteria content in the liver, spleen and brain organs of mice infected with 72h was significantly lower than that of the parent strain (P0.01). The invasiveness of MBMEC and RAW264.7 cells (P0.05) and the adhesion to MBMEC (P0.05), and the adhesion to SIEC (P0.05) were increased, but the intracellular proliferation difference was not significant. It was suggested that Lmo2714 and Lm were related to the pathogenicity of Lm, and could partly affect the virulence 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】：2017
【分类号】：S852.61

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