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
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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。
Bacteriophages are virus of bacteria.They are recognized to be the most numerous life-form in the biosphere.Since they were discoveried in 1915,bacteriophages have been at the forefront of molecular biological research,both as model systems and biological tools for the study and manipulation of bacterial genes.Many general concepts of contemporary biology were derived from work with phages.Lysogenic phages and the others mobile DNA elements(transposons,integrative plasmids,pathogenicity islands,and IS elements) are important vehicles for the gene lateral transfer between bacterial strains,then bring the biological diversity.They play an important role in bacteria evolution,antibiotic-resistant and pathogenicity,and also enhance the fitness of the bacteria.Deep insight into the genetic background of phages is very important for no matter investigating the interaction of phages and their host bacteria,and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phages.Currently,phage research is becoming a hot spot of microbiology.In addition,multidrug-resistant bacteria are becoming serious increasingly because of antibiotic abuse,and as the virus of infecting bacterias specifically, the bacteriophages in controlling bacterial infection are presented,some of which show therapeutic promise.Today,multivalency phages or engineering remolded phages have the optimistic perspective in antibacterial therapy.For this reason,it is very necessary to establish a method to remold phage genome.In this study,we focused on the biological characters of Pseudomonas aeruginosa phage PAP3,and remolded genome of Pseudomonas aeruginosa phage PaP3 through restriction endonucleasing,PCR deletion and religasing,at last,the remolded genome was electroporated into host cell.The contents and results are as follows:
1.The biological characters of PaP3.
①Electron microscopy revealed that the Pseudomonas aeruginosa phage PaP3 has an isometric head(about 65nm in diameter) and a short tail,and it belongs to pedoviridae families.
②Bacteriophage PaP3 is a temperate phage of Pseudomonas aeruginosa strain PA3, the plaques of PaP3 are semitransparent and their sizes are about 0.2cm in diameter.
③The one-step growth curve of PaP3 revealed that latent periods is about 20min,the rise periods is 60min,and the average burst size is about 31 phage particles per infected cell. The data demonstrate one life circle of PaP3 from phage absorption to offspring particle releasing.
④0.001 MOI-infected host bacteria is gained the highest phage offsprings,about 4.0×10~(10)pfu/ml.So the optimal multiplicity of infection of PaP3 is 0.001.
⑤Rate constant of reaction between antiserum and PaP3 is 262,it means that the antigenicity of PaP3 is moderate.Cross neutralization test among three phages indicates there is little dependablity among these three phages.
2.Genomic remolding ofPseudomonas aeruginosa phage PAP3.
PaP3 genomic DNA was cut by PacⅠ,SphⅠand SacⅡstep-by-step,and all the fragments were reclaimed using Gel extraction kit.The fragment contain tRNA genes was used as template to perform PCR deletion reaction.PCR product was cloned into pBSK vector and identified by sequencing.Finally,remolded genome ofPseudomonas aeruginosa phage PaP3 was obtained by ligating propotional fragment.
3.Tansforming remolded PaP3 genomic DNA into host cells by electroporation.
①Conditions of PaP3 genomic DNA electroporated into host cells.A P.aeruginosa strain PA3 was used as the recipient strain.Effects of growth stage of the strain, electroporation medium,Erythromycin concentration,osmotic pressure,field strength, DNA concentration and competent cells density on electroporation rate of PaP3 genomic DNA were observed under different conditions.It was showed that the highest transformation rate of electroporation was 2.1×10~3pfu/μgDNA under the condition in which the cells were cultured to stationary phase in LB adding 50μg/ml Erythromycin and concentrated to about 10~(11) cells/ml with 100mM sucrose at 25℃,the mixture of the competent cells and PaP3 DNA was eletroporated at 12kV/cm,300Ω,25μF.The results will be very helpful for the study of genomic function of Pseudomonas aeruginosa phage PAP3.
②Electroporation of remolded PaP3 genome.The rernolded genome DNA was electroporated under the condition above metioned,but none of infective phage was obtained finally.
In conclusion,the basic biological characters of Pseudomonas aeruginosa phage PaP3 was investigated,and the conditions of P.aeruginosa phage PaP3 genomic DNA electroporated into host cells was also explored.To study PaP3 tRNA genes function,four tRNA genes were deleted using PCR method.Though none of infective phage was obtained after remolded PaP3 genome had been electroporated,it will be a new approach to research genes function of phage.
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。
Bacteriophages are virus of bacteria.They are recognized to be the most numerous life-form in the biosphere.Since they were discoveried in 1915,bacteriophages have been at the forefront of molecular biological research,both as model systems and biological tools for the study and manipulation of bacterial genes.Many general concepts of contemporary biology were derived from work with phages.Lysogenic phages and the others mobile DNA elements(transposons,integrative plasmids,pathogenicity islands,and IS elements) are important vehicles for the gene lateral transfer between bacterial strains,then bring the biological diversity.They play an important role in bacteria evolution,antibiotic-resistant and pathogenicity,and also enhance the fitness of the bacteria.Deep insight into the genetic background of phages is very important for no matter investigating the interaction of phages and their host bacteria,and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phages.Currently,phage research is becoming a hot spot of microbiology.In addition,multidrug-resistant bacteria are becoming serious increasingly because of antibiotic abuse,and as the virus of infecting bacterias specifically, the bacteriophages in controlling bacterial infection are presented,some of which show therapeutic promise.Today,multivalency phages or engineering remolded phages have the optimistic perspective in antibacterial therapy.For this reason,it is very necessary to establish a method to remold phage genome.In this study,we focused on the biological characters of Pseudomonas aeruginosa phage PAP3,and remolded genome of Pseudomonas aeruginosa phage PaP3 through restriction endonucleasing,PCR deletion and religasing,at last,the remolded genome was electroporated into host cell.The contents and results are as follows:
1.The biological characters of PaP3.
①Electron microscopy revealed that the Pseudomonas aeruginosa phage PaP3 has an isometric head(about 65nm in diameter) and a short tail,and it belongs to pedoviridae families.
②Bacteriophage PaP3 is a temperate phage of Pseudomonas aeruginosa strain PA3, the plaques of PaP3 are semitransparent and their sizes are about 0.2cm in diameter.
③The one-step growth curve of PaP3 revealed that latent periods is about 20min,the rise periods is 60min,and the average burst size is about 31 phage particles per infected cell. The data demonstrate one life circle of PaP3 from phage absorption to offspring particle releasing.
④0.001 MOI-infected host bacteria is gained the highest phage offsprings,about 4.0×10~(10)pfu/ml.So the optimal multiplicity of infection of PaP3 is 0.001.
⑤Rate constant of reaction between antiserum and PaP3 is 262,it means that the antigenicity of PaP3 is moderate.Cross neutralization test among three phages indicates there is little dependablity among these three phages.
2.Genomic remolding ofPseudomonas aeruginosa phage PAP3.
PaP3 genomic DNA was cut by PacⅠ,SphⅠand SacⅡstep-by-step,and all the fragments were reclaimed using Gel extraction kit.The fragment contain tRNA genes was used as template to perform PCR deletion reaction.PCR product was cloned into pBSK vector and identified by sequencing.Finally,remolded genome ofPseudomonas aeruginosa phage PaP3 was obtained by ligating propotional fragment.
3.Tansforming remolded PaP3 genomic DNA into host cells by electroporation.
①Conditions of PaP3 genomic DNA electroporated into host cells.A P.aeruginosa strain PA3 was used as the recipient strain.Effects of growth stage of the strain, electroporation medium,Erythromycin concentration,osmotic pressure,field strength, DNA concentration and competent cells density on electroporation rate of PaP3 genomic DNA were observed under different conditions.It was showed that the highest transformation rate of electroporation was 2.1×10~3pfu/μgDNA under the condition in which the cells were cultured to stationary phase in LB adding 50μg/ml Erythromycin and concentrated to about 10~(11) cells/ml with 100mM sucrose at 25℃,the mixture of the competent cells and PaP3 DNA was eletroporated at 12kV/cm,300Ω,25μF.The results will be very helpful for the study of genomic function of Pseudomonas aeruginosa phage PAP3.
②Electroporation of remolded PaP3 genome.The rernolded genome DNA was electroporated under the condition above metioned,but none of infective phage was obtained finally.
In conclusion,the basic biological characters of Pseudomonas aeruginosa phage PaP3 was investigated,and the conditions of P.aeruginosa phage PaP3 genomic DNA electroporated into host cells was also explored.To study PaP3 tRNA genes function,four tRNA genes were deleted using PCR method.Though none of infective phage was obtained after remolded PaP3 genome had been electroporated,it will be a new approach to research genes function of phage.
引文
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