红平红球菌复苏促进因子基因克

发布时间：2018-06-16 07:29

  本文选题：红平红球菌 + 非可培养　； 参考：《兰州理工大学》2017年硕士论文

【摘要】：细菌活的非可培养(VBNC)状态是细菌在不良环境下形成的一种休眠方式。在所在环境改善时非可培养细胞复苏为可培养状态,但有关细菌复苏机制并不明确。藤黄微球菌(Micrococcus luteus)细胞复苏促进因子(Resuscitation promoting factor,Rpf)首次被报道能促使休眠菌状态细胞复苏,并刺激正常细胞生长。现已发现复苏促进因子(Rpf)广泛存在于高G+C含量革兰氏阳性细菌中,与细菌生长及抵御不良环境有关。不同种类细菌的Rpf种类和数量有一定差异。红球菌属的细菌广泛分布于岩石、土壤、地下水等自然环境。多数种类在修复石油污染土壤、污水处理等方面有有广阔应用前景。红平红球菌(Rhodococcus erythropolis)KB1分离自石油污染土壤,具有高效石油降解能力和环境适应性。为探索Rpf在红平红球菌适应环境中的作用,本文进行了红平红球菌KB1 Rpf基因克隆、基因结构分析、表达及生物学性质研究,具体内容如下:设计了复苏促进因子基因特异引物,从红平红球菌KB1基因组DNA扩增出4种rpf基因,分别为564、1125、1128和555 bp,编码187、353、375和184个氨基酸残基的蛋白质。序列分析发现4种rpf基因分别与藤黄微球菌rpf基因,结核分枝杆菌(Mycobacterium tuberculosis)rpf B、rpf C及链霉菌(Streptomyces coelicolor)rpf B有较高相似性。4个rpf基因编码的蛋白都有一个类似藤黄微球菌细胞复苏促进因子的Rpf域,含有1个保守谷氨酸残基,由大约70个氨基酸组成。其中Rpf-1与结核分枝杆菌、链霉菌Rpf C相似,只含1个Rpf结构域,Rpf-2与结核分枝杆菌Rpf B相似,含有1个Rpf域、1个G5域和2个DUF348,Rpf-3与链霉菌Rpf B相似,含有1个Rpf域、G5域和3个DUF348,Rpf-4与藤黄微球菌Rpf相似,含1个Rpf域和1个Lys M域。Rpf-2、Rpf-3含信号肽序列,Rpf-1、Rpf-4不含信号肽。Rpf-1、Rpf-2、Rpf-3和Rpf-4均为亲水性蛋白,含有多个丝氨酸、苏氨酸和酪氨酸位点,没有组氨酸位点。设计特异表达引物,将红平红球菌KB1 rpf-1基因克隆于原核表达载体p ET-32a(+),在大肠杆菌BL21中高效表达,用Ni琼脂糖亲和层析柱纯化重组蛋白,SDS-PAGE电泳分析表明纯化蛋白分子量为36k Da。用人工合成底物4-Methylumbelliferyl-β-D-N-N’-N’-triacetyl chitotriose检测发现rpf-1具有溶菌酶活性。溶菌酶比活力为2.07 U/mg。以偶氮酪蛋白为底物测定了纯化蛋白rpf-1的蛋白酶活性,发现纯化蛋白能水解偶氮酪蛋白,蛋白水解酶比活力为235 U/mg。将纯化的重组蛋白Rpf-1添加到不同状态红平红球菌细胞培养物中,发现其对正常生长、VBNC状态及-80℃低温保存的细胞具有明显复苏或促生长作用。添加10%的Rpf-1使低温保存红平红球菌生长速度增长4.8-6倍。添加Rpf-1使正常生长的细菌细胞在12 h时入对数期,48 h细菌数量达最大,是对照组细菌生长量的3倍。添加Rpf-1重组蛋白使VBNC状态的红平红球菌复苏量增加,48 h后细菌生长量是空白对照组的1.44倍。
[Abstract]:The non-culturable VBNCstate of bacteria is a dormant way for bacteria to form in bad environment. The resuscitation of non-culturable cells was culturable when the environment was improved, but the mechanism of bacterial resuscitation was not clear. It was reported for the first time that Micrococcus luteus could promote the recovery of dormancy cells and stimulate the growth of normal cells. It has been found that the resuscitation promoter rpf) is widely found in Gram-positive bacteria with high G C content, which is related to bacterial growth and resistance to adverse environment. The species and quantity of RPF of different bacteria are different to some extent. Rhodococcus bacteria are widely distributed in rock, soil, groundwater and other natural environment. Most species have broad application prospects in oil contaminated soil remediation, sewage treatment and so on. Rhodococcus erythropolisus KB1 was isolated from petroleum contaminated soil and has high oil degradation ability and environmental adaptability. In order to explore the role of RPF in the adaptive environment of Rhodococcus hongpingensis, the gene cloning, gene structure analysis, expression and biological properties of KB1 Rpf gene were studied. The main contents were as follows: a specific primer for resuscitation promoting factor gene was designed. Four rpf genes encoding 187353375 and 184 amino acid residues were amplified from the KB1 genome of Rhodococcus rubrum. Sequence analysis revealed that four rpf genes were associated with rpf gene of Micrococcus lutei. Mycobacterium tuberculosis)rpf rpfC and Streptomyces coelicolor)rpf B have high similarity. The four rpf gene encoded proteins have a RPF domain similar to micrococcus luteum resuscitation factor, and contain a conserved glutamate residue. It consists of about 70 amino acids. Among them, Rpf-1 is similar to Mycobacterium tuberculosis, StrepfC, only one RPF domain Rpf-2 is similar to Mycobacterium tuberculosis RpfB, and one RPF domain, one G5 domain and two DUF348Rpf-3 are similar to Strepfella rpf-3. The Rpf-4 containing one RPF domain G5 and three DUF348C Rpf-4 is similar to Rpf of Micrococcus luteum, and contains one RPF domain and one Lys M domain. Rpf-2N Rpf-3 contains no signal peptide. Rpf-1Rpf-2 rpf-3 and Rpf-4 are hydrophilic proteins, and contain multiple serine, threonine and tyrosine sites, and Rpf-4 have multiple serine, threonine and tyrosine sites, and Rpf-1, Rpf-2, Rpf-3 and Rpf-4 are hydrophilic proteins with multiple serine, threonine and tyrosine sites. There is no histidine site. A specific expression primer was designed and cloned into the prokaryotic expression vector pET-32a (pET-32a). The recombinant protein was purified by Ni agarose affinity chromatography. The molecular weight of the purified protein was 36kDa. The synthetic substrates 4-Methylumbelliferyl- 尾 -D-N-Na-Na-N-triacetyl chitotriose showed that rpf-1 had lysozyme activity. The specific activity of lysozyme was 2.07 U / mg. The protease activity of purified protein rpf-1 was determined by using azo casein as substrate. It was found that the purified protein could hydrolyze azo casein and the specific activity of protein hydrolase was 235U / mg. The purified recombinant protein Rpf-1 was added to different cultures of Rhodococcus erythropoides. It was found that Rpf-1 could significantly resuscitate or promote the growth of normal growth cells in VBNC state and cryopreserved at -80 鈩，

本文编号：2025899