本文選題：Lm + 蛋白組學�。� 參考：《石河子大學》2017年碩士論文

【摘要】：單核細胞增多性李斯特菌(listeria monocytogenes,Lm)是能夠穿越人和動物三大屏障從而引起人和動物發(fā)病的一種食源性致病菌,主要引起敗血癥、胃腸炎、腦膜炎等臨床癥狀,Lm的發(fā)病率不高,但死亡率較高。在Lm細胞胞壁上存在許多與細菌毒力密切相關的表面蛋白,其中有一類蛋白,羧基端帶有LPXTG基序,并且被分選酶A(Sortase A,Srt A)識別,通過共價的方式錨定到肽聚糖上,呈現在細菌表面。本研究以臨床分離株LM90SB2(綿羊腦組織中單核細胞增多性李斯特菌,4b血清型)為研究對象,利用蛋白組學技術對LM90SB2和Srt A缺失株的細胞壁蛋白進行差異蛋白的篩選,運用生物信息學分析差異蛋白可能的功能;對細胞壁表面蛋白Lmo2714構建缺失株,研究其在細胞粘附與侵襲,小鼠毒力試驗,環(huán)境適應性等生物學特性中的作用,為挖掘新的細胞壁表面蛋白以及致病機制奠定基礎。1.變溶菌素(Mutanolysin)酶解Lm細胞壁最佳條件的篩選:為了提高酶解法結合TCA-丙酮沉淀法提取臨床分離株LB90SB2細胞壁表面蛋白的效率,本試驗對變溶菌素的濃度及酶解時間進行了優(yōu)化。選擇20μg/mL,60μg/mL的變溶菌素濃度進行30min-4 h,37℃酶解條件及37℃和4℃低溫聯(lián)合酶解的條件,通過測定酶解前后OD600、CFU的變化,以此判斷細胞膜的完整性和計算原生質體形成率,篩選出最佳酶解濃度及酶解時間的條件。在酶濃度的最佳條件下,再測定不同酶解時間段細胞壁蛋白的濃度再測定不同酶解時間段細胞壁蛋白提取濃度。結果表明,在酶解30 min-4 h時間段內,原生質體形成率可以從0提高到91.00%,細胞壁蛋白濃度從0.272 mg/mL增加到1.735mg/mL。△OD600在1.86%-14.50%變動,但△OD600的統(tǒng)計學處理差異不顯著。變溶菌素濃度在60μg/m L時,作用4 h,原生質體的形成率能夠達到91.00%,遠遠高于20μg/mL的變溶菌素同樣作用4 h的78%。最終,60μg/mL為變溶菌素濃度,4h的酶解時間,是酶解LM90SB2細胞壁的最佳條件。這為下一步研究細胞壁蛋白篩選奠定重要基礎。2.LM90SB2與LM90SB2 srtA基因缺失株(LM90SB2-△srtA)細胞壁差異蛋白篩選:為進一步探明由srtA分選的細胞壁表面蛋白譜,本試驗采用非標記定量技術(Lable-free)結合液相色譜-串聯(lián)質譜(LC-MS/MS)技術篩選差異蛋白,通過生物信息學分析,對差異蛋白進行功能預測。結果顯示,利用非標記定量技術結合LC-MS/MS,共檢測出蛋白組數697個,肽段數3367個,差異顯著蛋白36個,其中20個蛋白在親本株中檢測到,在缺失株未檢出;7個蛋白在缺失株檢測到的,在親本株未檢出;7個蛋白在親本株中的蛋白量大于缺失株(P0.05),2個蛋白在親本株中的蛋白量小于缺失株(P0.05);生物信息學分析,這36個差異顯著蛋白與內化素、細胞壁錨定蛋白等有關。srtA分選蛋白譜的探明,為挖掘新的毒力因子或藥物靶標奠定了基礎。3.LM90SB2-△lmo2714缺失株的構建:為成功構建LM90SB2-△lmo2714,本試驗利用同源重組技術。首先設計lmo2714基因上下臂特異性引物,通過PCR技術擴增,接著通過SOE-PCR技術得到lmo2714基因的缺失片段即△lmo2714,然后與pMD19-T連接,將測序正確的△lmo2714再與pKSV7連接,pKSV7-△lmo2714電轉化LM90SB2感受態(tài)細胞中,陽性轉化子在高溫和氯霉素抗性的雙重壓力條件下連續(xù)傳代,使之發(fā)生同源重組,同時用旁外側引物(lmo2714 dele-1和lmo2714 dele-2)PCR檢測缺失片段,最后置于無抗性的30℃條件下獲得無質粒的LM90SB2-△lmo2714,連續(xù)傳代后檢測缺失株穩(wěn)定性。結果顯示:利用旁側引物檢測構建的缺失株,可以觀察到大小為1033bp的條帶,而親本株的條帶大小為1981bp,盲傳20代之后,沒有發(fā)生返祖現象。表明成功構建LM90SB2-△lmo2714缺失株且遺傳穩(wěn)定性表現良好。4.LM90SB2-△lmo2714缺失株的生物學特性:為探究Lmo2714的功能,將親本株作為對照,檢測菌株的生物膜形成能力、生化特性、對環(huán)境的適應性、對小鼠的致病性及對SIEC,MBMEC,RAW264.7和HBMEC的粘附、侵襲能力及胞內增殖情況。結果顯示:缺失株對環(huán)境的適應性以及生物膜形成能力沒有明顯差異;影響部分生化特性;但LM90SB2-△lmo2714小鼠LD50比親本株高1.34個對數級(P0.05),感染小鼠72h后的肝、脾、腦臟器中的含菌量極顯著低于親本株(P0.01);顯著降低了對MBMEC和RAW264.7細胞的侵襲能力(P0.05)以及對MBMEC的粘附能力(P0.05);提高了對SIEC的粘附能力(P0.05);但是胞內增殖差異不顯著。推測Lmo2714與Lm的致病性有一定關系,能部分影響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.

【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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