铜绿假单胞菌噬菌体PaP3生物学特性的研究与噬菌体基因组改造的技术准备
发布时间：2019-06-24 17:49
【摘要】： 噬菌体是细菌的病毒,广泛的存在于自然环境之中。作为细菌之间基因水平转移的重要载体,噬菌体与转座子、整合型质粒、致病岛、插入序列等可移动DNA元件一起,在细菌基因组的进化、细菌的致病性、耐药性以及细菌对环境的适应性中都具有非常关键的作用。溶原性噬菌体通过自身携带的外源性遗传物质改变着宿主菌及自身的基因组构成,进而改变了宿主菌的生物学性状,对噬菌体展开广泛深入的研究对于了解噬菌体与宿主的相互作用、揭示生物的多样性等方面具有重要的意义。因此,噬菌体及其基因组功能的研究成为微生物学研究的热点领域之一。近年来由于抗生素的广泛使用,细菌的多重耐药现象日益严重,寻求新的抗菌手段已成为当务之急。噬菌体作为特异性感染并裂解细菌的病毒,可望成为一种新的抗细菌感染制剂。但是,噬菌体感染细菌的特异性决定了它的抗菌谱很窄,为了解决这一问题,必须对噬菌体进行人工改造以扩大它的宿主谱。在分子生物学技术高度发展的今天,人工改造噬菌体已经成为可能。但在进行噬菌体改造之前,必须进行一些必要的技术准备,如:通过什么手段来改造噬菌体,改造后的噬菌体基因组如何导入宿主菌从而产生感染性的噬菌体颗粒等等。这些都是有待于克服的技术屏障。 本文以本室分离鉴定的一株铜绿假单胞菌噬菌体PaP3为研究对象,首先探讨其生物学特性;进而建立一种利用分子生物学技术改造噬菌体基因组的方法;最后探讨将改造后的噬菌体基因组电转导入宿主菌的方法。研究内容及其结果主要包括以下几个方面: 1.铜绿假单胞菌噬菌体PaP3生物学特性的研究。 ①电镜观察发现,PaP3有一个多面体立体对称头部,头部直径约为65nm,无囊膜,有一个短尾。从病毒颗粒的形态来看,噬菌体PaP3属于短尾噬菌体科。 ②PaP3感染其宿主菌铜绿假单胞菌PA3形成的噬斑明显,为圆形半透明,直径在2mm左右,呈典型的温和噬菌体的噬斑特征。 ③测定噬菌体PaP3的一步生长曲线,得知PaP3感染宿主菌的潜伏期约为20min,爆发期约为60min,爆发量约为31。该曲线反映出噬菌体从感染宿主菌到子代释放的一个完整的生活周期。 ④当MOI=0.001时,噬菌体PaP3感染其宿主菌产生的子代噬菌体滴度为4.0×10~(10)pfu/ml,产出率最高。确定了噬菌体PaP3感染其宿主菌铜绿假单胞菌的最佳感染复数为0.001。 ⑤测定了本室保存的三株铜绿假单胞菌噬菌体与抗血清之间的交叉吸附常数。结果表明,三株噬菌体的抗血清只抑制自身与对应宿主菌的结合,不存在交叉抑制吸附关系。说明三株噬菌体的吸附结构之间不存在相关性。 2.噬菌体PaP3基因组的改造 基因组的改造包括基因的插入、删除、定点突变等方式。本研究采用删除目的基因的方法对PaP3基因组进行改造,即选择限制性内切酶PacⅠ、SphⅠ和SacⅡ对PaP3基因组进行分步酶切,获得目的基因所在的片段,再利用PCR删除技术去除该目的基因(在本研究中为tRNA基因)。最后将删除改造后的片段与其他酶切片段按顺序连接起来,即获得人工改造后的PaP3基因组。 3.改造后PaP3基因组电转化宿主菌。 ①PaP3基因组电转化宿主菌的条件摸索。纯化噬菌体PaP3完整基因组DNA,以铜绿假单胞菌(Pseudomonas aeruginosa)PA3为受体菌,探讨电转化基本条件,包括:细胞生长状态、感受态细胞的制备方式、电场强度、DNA浓度、细胞密度等条件对转化效率的影响。确定了一组适用于电转化噬菌体PaP3基因组DNA的条件:在含50μg/ml红霉素的LB培养基中培养宿主菌14h—16h,以100mM蔗糖溶液为介质,在25℃条件下制备感受态细胞,适宜的感受态细胞浓度为10~(11)/ml,适宜的电转参数为12kV/cm,300Ω,25μF。在此条件下获得较高的转化效率,可达2.1×10~3pfu/μg DNA。这为改造后噬菌体基因组的电转奠定了基础。 ②改造后基因组的电转。将删除了tRNA基因的PaP3基因组按前面摸索的铜绿假单胞菌噬菌体基因组电转化宿主菌的条件进行电转,未能获得具有感染性的噬菌体颗粒。在本文中对其原因进行了探讨。 综上所述,通过本研究,初步明确了铜绿假单胞菌噬菌体PaP3的基本生物学特性,并探讨了其基因组电转化宿主菌并获得具有感染性的噬菌体颗粒的基本条件,同时通过PCR删除技术去除基因组中的tRNA基因,并将改造后的噬菌体DNA再次转入宿主菌体内,以期了解噬菌体tRNA基因的功能。最后虽然没有获得改造后的噬菌体颗粒,但为我们研究噬菌体基因功能以及对噬菌体进行人工改造进行了技术上的探索。
[Abstract]:Phage is a virus of bacteria and is widely present in the natural environment. As an important carrier for gene level transfer between bacteria, the phage is combined with a movable DNA element such as a transposon, a whole-type plasmid, a disease-causing island, an insertion sequence and the like, The drug resistance and the bacteria have a very critical role in the adaptation of the environment. The lysogenic bacteriophage changes the host bacteria and its own genome by the exogenous genetic material carried by itself, thus the biological character of the host bacterium is changed, and the extensive and in-depth research on the phage is used to understand the interaction between the bacteriophage and the host, It is of great significance to reveal the biological diversity and so on. Therefore, the research of the function of the phage and its genome has become one of the hot spots in the study of microbiology. In recent years, because of the wide use of antibiotics, the multi-drug resistance of bacteria is becoming more and more serious, and the search for new anti-bacterial means has become an urgent task. Phage as a virus that specifically infects and lyses bacteria is expected to be a new anti-bacterial infection. However, the specificity of the phage-infected bacteria determines that its antibacterial spectrum is very narrow. In order to solve this problem, the phage must be artificially modified to expand its host spectrum. The artificial transformation of the phage has become possible today in the highly developed molecular biology technology. But before the phage transformation, some necessary technical preparations must be made, such as by means of the transformation of the phage, how the transformed phage genome is introduced into the host strain to produce infectious bacteriophage particles, and the like. These are the technical barriers to be overcome. In this paper, a strain of P. aeruginosa phage PaP3 isolated and identified in this room was used as a research object, and its biological characteristics were first discussed. Methods: The transformation of the transformed phage genome into the host strain was discussed. Methods: The contents and results of the study mainly include the following: Aspect:1. Pseudomonas aeruginosa Phage PaP3 Biology The study of the characteristics of PaP3 showed that PaP3 had a three-dimensional symmetric head with a head diameter of about 65 nm. The capsule, with a short tail. From the morphology of the virus particles, the phage PaP3 belonging to the short-tail phage family. The plaque formed by the Pseudomonas aeruginosa PA3 of the host strain of the pPaP3 infection is obvious, is circular and semi-transparent, has a diameter of about 2 mm, The one-step growth curve of the phage PaP3 was determined, and the incubation period of PaP3 infected host bacteria was about 20 min, and the outbreak period was about 20 min. For 60 min, the amount of outbreak was about 31. The curve reflects the infection of the phage from the infected host to the host. A complete cycle of life for the release of the progeny. When MOI = 0.001, the phage PaP3 infects its host strain with a progeny phage titer of 4.0 to 10 ~ (10) pfu/ ml, and the yield was the highest. It was determined that the phage PaP3 was infected with its host bacteria, P. aeruginosa. The best infection number of Pseudomonas aeruginosa is 0.001. The amount of the three P. aeruginosa preserved in this room is determined by the method of the present invention. The cross-adsorption constant between the phage and the antisera of the pseudomonads showed that the antisera of Sanzhu phage only inhibited the autoantibodies. The binding of the corresponding host bacteria does not have a cross-inhibiting adsorption relationship. There is no difference between the adsorption structure of the three phage. There is a correlation.2. The transformation of the genome of the phage PaP3 The transformation of the genome includes the insertion, deletion and site-directed mutagenesis of the gene. The method of deleting the target gene is used to transform the PaP3 genome, i.e., the restriction enzyme Pac I, Sph I and Sac II are selected. carrying out step-by-step enzyme digestion on the PaP3 genome to obtain a fragment of the target gene, and then using a PC; R deletion technique removes the target gene (tRNA gene in this study). Finally, the modified fragment is removed from other enzymes the segments are connected in sequence, i. e., after an artificial modification is obtained, PaP3 genome.3. PaP3 after modification The genomic DNA of the genome is transformed into the host strain. The genomic DNA of the PaP3 genome is purified. The complete genomic DNA of the phage PaP3 is purified, and the Pseudomonas aeruginosa PA3 is the acceptor, and the basic conditions of the electric transformation are discussed, including the cell growth state and the competent cell. The effect of the conditions of the preparation method, the electric field intensity, the concentration of DNA, the cell density and the like on the conversion efficiency was determined. A set of conditions suitable for the genomic DNA of the electric-transformed phage PaP3 were determined. The host bacteria were cultured for 14 h and 16 h in LB medium containing 50. m u.g/ ml of erythromycin, and the medium was taken as a medium at 25.degree. C. with a 100 mM sucrose solution. The competent cells were prepared under the conditions of 10 to (11)/ ml, and the appropriate electrorotation parameters were 12 kV/ cm,300 惟,25. m high conversion efficiency under the condition of up to 2.1%1 0-3pfu/ ug DN A. This provides a basis for the electrorotation of the post-engineered phage genome. The transformation of the genome after the transformation. The PaP3 genome of the tRNA gene is deleted. the strip of the host bacteria for the transformation of the host bacteria by the phage genome In the light of the above, the basic biological characteristics of P. aeruginosa phage PaP3 were identified, and the host bacteria of the genome of Pseudomonas aeruginosa were also discussed. the basic conditions of the infected phage particles, while the tRNA gene in the genome is removed by the PCR deletion technique, and the modified phage DNA is transferred to the host bacterium again, so as to know the function of the bacteriophage tRNA gene, and finally, the modified phage DNA is not obtained,
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378;R516
本文编号：2505247
[Abstract]:Phage is a virus of bacteria and is widely present in the natural environment. As an important carrier for gene level transfer between bacteria, the phage is combined with a movable DNA element such as a transposon, a whole-type plasmid, a disease-causing island, an insertion sequence and the like, The drug resistance and the bacteria have a very critical role in the adaptation of the environment. The lysogenic bacteriophage changes the host bacteria and its own genome by the exogenous genetic material carried by itself, thus the biological character of the host bacterium is changed, and the extensive and in-depth research on the phage is used to understand the interaction between the bacteriophage and the host, It is of great significance to reveal the biological diversity and so on. Therefore, the research of the function of the phage and its genome has become one of the hot spots in the study of microbiology. In recent years, because of the wide use of antibiotics, the multi-drug resistance of bacteria is becoming more and more serious, and the search for new anti-bacterial means has become an urgent task. Phage as a virus that specifically infects and lyses bacteria is expected to be a new anti-bacterial infection. However, the specificity of the phage-infected bacteria determines that its antibacterial spectrum is very narrow. In order to solve this problem, the phage must be artificially modified to expand its host spectrum. The artificial transformation of the phage has become possible today in the highly developed molecular biology technology. But before the phage transformation, some necessary technical preparations must be made, such as by means of the transformation of the phage, how the transformed phage genome is introduced into the host strain to produce infectious bacteriophage particles, and the like. These are the technical barriers to be overcome. In this paper, a strain of P. aeruginosa phage PaP3 isolated and identified in this room was used as a research object, and its biological characteristics were first discussed. Methods: The transformation of the transformed phage genome into the host strain was discussed. Methods: The contents and results of the study mainly include the following: Aspect:1. Pseudomonas aeruginosa Phage PaP3 Biology The study of the characteristics of PaP3 showed that PaP3 had a three-dimensional symmetric head with a head diameter of about 65 nm. The capsule, with a short tail. From the morphology of the virus particles, the phage PaP3 belonging to the short-tail phage family. The plaque formed by the Pseudomonas aeruginosa PA3 of the host strain of the pPaP3 infection is obvious, is circular and semi-transparent, has a diameter of about 2 mm, The one-step growth curve of the phage PaP3 was determined, and the incubation period of PaP3 infected host bacteria was about 20 min, and the outbreak period was about 20 min. For 60 min, the amount of outbreak was about 31. The curve reflects the infection of the phage from the infected host to the host. A complete cycle of life for the release of the progeny. When MOI = 0.001, the phage PaP3 infects its host strain with a progeny phage titer of 4.0 to 10 ~ (10) pfu/ ml, and the yield was the highest. It was determined that the phage PaP3 was infected with its host bacteria, P. aeruginosa. The best infection number of Pseudomonas aeruginosa is 0.001. The amount of the three P. aeruginosa preserved in this room is determined by the method of the present invention. The cross-adsorption constant between the phage and the antisera of the pseudomonads showed that the antisera of Sanzhu phage only inhibited the autoantibodies. The binding of the corresponding host bacteria does not have a cross-inhibiting adsorption relationship. There is no difference between the adsorption structure of the three phage. There is a correlation.2. The transformation of the genome of the phage PaP3 The transformation of the genome includes the insertion, deletion and site-directed mutagenesis of the gene. The method of deleting the target gene is used to transform the PaP3 genome, i.e., the restriction enzyme Pac I, Sph I and Sac II are selected. carrying out step-by-step enzyme digestion on the PaP3 genome to obtain a fragment of the target gene, and then using a PC; R deletion technique removes the target gene (tRNA gene in this study). Finally, the modified fragment is removed from other enzymes the segments are connected in sequence, i. e., after an artificial modification is obtained, PaP3 genome.3. PaP3 after modification The genomic DNA of the genome is transformed into the host strain. The genomic DNA of the PaP3 genome is purified. The complete genomic DNA of the phage PaP3 is purified, and the Pseudomonas aeruginosa PA3 is the acceptor, and the basic conditions of the electric transformation are discussed, including the cell growth state and the competent cell. The effect of the conditions of the preparation method, the electric field intensity, the concentration of DNA, the cell density and the like on the conversion efficiency was determined. A set of conditions suitable for the genomic DNA of the electric-transformed phage PaP3 were determined. The host bacteria were cultured for 14 h and 16 h in LB medium containing 50. m u.g/ ml of erythromycin, and the medium was taken as a medium at 25.degree. C. with a 100 mM sucrose solution. The competent cells were prepared under the conditions of 10 to (11)/ ml, and the appropriate electrorotation parameters were 12 kV/ cm,300 惟,25. m high conversion efficiency under the condition of up to 2.1%1 0-3pfu/ ug DN A. This provides a basis for the electrorotation of the post-engineered phage genome. The transformation of the genome after the transformation. The PaP3 genome of the tRNA gene is deleted. the strip of the host bacteria for the transformation of the host bacteria by the phage genome In the light of the above, the basic biological characteristics of P. aeruginosa phage PaP3 were identified, and the host bacteria of the genome of Pseudomonas aeruginosa were also discussed. the basic conditions of the infected phage particles, while the tRNA gene in the genome is removed by the PCR deletion technique, and the modified phage DNA is transferred to the host bacterium again, so as to know the function of the bacteriophage tRNA gene, and finally, the modified phage DNA is not obtained,
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378;R516
【参考文献】
相关期刊论文 前3条
1 李明,申晓冬,周莹冰,黄建军,胡晓梅,饶贤才,胡福泉;铜绿假单胞菌噬菌体PaP1生物学特性的研究[J];第三军医大学学报;2005年09期
2 黄建军,胡晓梅,饶贤才,张克斌,金晓琳,周莹冰,李明,申晓冬,朱军民,胡福泉;铜绿假单胞菌噬菌体PaP2生物学特性的研究[J];第三军医大学学报;2004年13期
3 张克斌,陈志瑾,金晓琳,饶贤才,胡福泉;铜绿假单胞菌噬菌体的分离鉴定及耐噬菌体突变频率测定[J];微生物学通报;2002年01期
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