銅綠假單胞菌噬菌體PaP3生物學(xué)特性的研究與噬菌體基因組改造的技術(shù)準(zhǔn)備
發(fā)布時(shí)間：2019-06-24 17:49
【摘要】： 噬菌體是細(xì)菌的病毒,廣泛的存在于自然環(huán)境之中。作為細(xì)菌之間基因水平轉(zhuǎn)移的重要載體,噬菌體與轉(zhuǎn)座子、整合型質(zhì)粒、致病島、插入序列等可移動(dòng)DNA元件一起,在細(xì)菌基因組的進(jìn)化、細(xì)菌的致病性、耐藥性以及細(xì)菌對(duì)環(huán)境的適應(yīng)性中都具有非常關(guān)鍵的作用。溶原性噬菌體通過自身攜帶的外源性遺傳物質(zhì)改變著宿主菌及自身的基因組構(gòu)成,進(jìn)而改變了宿主菌的生物學(xué)性狀,對(duì)噬菌體展開廣泛深入的研究對(duì)于了解噬菌體與宿主的相互作用、揭示生物的多樣性等方面具有重要的意義。因此,噬菌體及其基因組功能的研究成為微生物學(xué)研究的熱點(diǎn)領(lǐng)域之一。近年來由于抗生素的廣泛使用,細(xì)菌的多重耐藥現(xiàn)象日益嚴(yán)重,尋求新的抗菌手段已成為當(dāng)務(wù)之急。噬菌體作為特異性感染并裂解細(xì)菌的病毒,可望成為一種新的抗細(xì)菌感染制劑。但是,噬菌體感染細(xì)菌的特異性決定了它的抗菌譜很窄,為了解決這一問題,必須對(duì)噬菌體進(jìn)行人工改造以擴(kuò)大它的宿主譜。在分子生物學(xué)技術(shù)高度發(fā)展的今天,人工改造噬菌體已經(jīng)成為可能。但在進(jìn)行噬菌體改造之前,必須進(jìn)行一些必要的技術(shù)準(zhǔn)備,如:通過什么手段來改造噬菌體,改造后的噬菌體基因組如何導(dǎo)入宿主菌從而產(chǎn)生感染性的噬菌體顆粒等等。這些都是有待于克服的技術(shù)屏障。 本文以本室分離鑒定的一株銅綠假單胞菌噬菌體PaP3為研究對(duì)象,首先探討其生物學(xué)特性;進(jìn)而建立一種利用分子生物學(xué)技術(shù)改造噬菌體基因組的方法;最后探討將改造后的噬菌體基因組電轉(zhuǎn)導(dǎo)入宿主菌的方法。研究?jī)?nèi)容及其結(jié)果主要包括以下幾個(gè)方面: 1.銅綠假單胞菌噬菌體PaP3生物學(xué)特性的研究。 ①電鏡觀察發(fā)現(xiàn),PaP3有一個(gè)多面體立體對(duì)稱頭部,頭部直徑約為65nm,無囊膜,有一個(gè)短尾。從病毒顆粒的形態(tài)來看,噬菌體PaP3屬于短尾噬菌體科。 ②PaP3感染其宿主菌銅綠假單胞菌PA3形成的噬斑明顯,為圓形半透明,直徑在2mm左右,呈典型的溫和噬菌體的噬斑特征。 ③測(cè)定噬菌體PaP3的一步生長(zhǎng)曲線,得知PaP3感染宿主菌的潛伏期約為20min,爆發(fā)期約為60min,爆發(fā)量約為31。該曲線反映出噬菌體從感染宿主菌到子代釋放的一個(gè)完整的生活周期。 ④當(dāng)MOI=0.001時(shí),噬菌體PaP3感染其宿主菌產(chǎn)生的子代噬菌體滴度為4.0×10~(10)pfu/ml,產(chǎn)出率最高。確定了噬菌體PaP3感染其宿主菌銅綠假單胞菌的最佳感染復(fù)數(shù)為0.001。 ⑤測(cè)定了本室保存的三株銅綠假單胞菌噬菌體與抗血清之間的交叉吸附常數(shù)。結(jié)果表明,三株噬菌體的抗血清只抑制自身與對(duì)應(yīng)宿主菌的結(jié)合,不存在交叉抑制吸附關(guān)系。說明三株噬菌體的吸附結(jié)構(gòu)之間不存在相關(guān)性。 2.噬菌體PaP3基因組的改造 基因組的改造包括基因的插入、刪除、定點(diǎn)突變等方式。本研究采用刪除目的基因的方法對(duì)PaP3基因組進(jìn)行改造,即選擇限制性內(nèi)切酶PacⅠ、SphⅠ和SacⅡ?qū)aP3基因組進(jìn)行分步酶切,獲得目的基因所在的片段,再利用PCR刪除技術(shù)去除該目的基因(在本研究中為tRNA基因)。最后將刪除改造后的片段與其他酶切片段按順序連接起來,即獲得人工改造后的PaP3基因組。 3.改造后PaP3基因組電轉(zhuǎn)化宿主菌。 ①PaP3基因組電轉(zhuǎn)化宿主菌的條件摸索。純化噬菌體PaP3完整基因組DNA,以銅綠假單胞菌(Pseudomonas aeruginosa)PA3為受體菌,探討電轉(zhuǎn)化基本條件,包括:細(xì)胞生長(zhǎng)狀態(tài)、感受態(tài)細(xì)胞的制備方式、電場(chǎng)強(qiáng)度、DNA濃度、細(xì)胞密度等條件對(duì)轉(zhuǎn)化效率的影響。確定了一組適用于電轉(zhuǎn)化噬菌體PaP3基因組DNA的條件:在含50μg/ml紅霉素的LB培養(yǎng)基中培養(yǎng)宿主菌14h—16h,以100mM蔗糖溶液為介質(zhì),在25℃條件下制備感受態(tài)細(xì)胞,適宜的感受態(tài)細(xì)胞濃度為10~(11)/ml,適宜的電轉(zhuǎn)參數(shù)為12kV/cm,300Ω,25μF。在此條件下獲得較高的轉(zhuǎn)化效率,可達(dá)2.1×10~3pfu/μg DNA。這為改造后噬菌體基因組的電轉(zhuǎn)奠定了基礎(chǔ)。 ②改造后基因組的電轉(zhuǎn)。將刪除了tRNA基因的PaP3基因組按前面摸索的銅綠假單胞菌噬菌體基因組電轉(zhuǎn)化宿主菌的條件進(jìn)行電轉(zhuǎn),未能獲得具有感染性的噬菌體顆粒。在本文中對(duì)其原因進(jìn)行了探討。 綜上所述,通過本研究,初步明確了銅綠假單胞菌噬菌體PaP3的基本生物學(xué)特性,并探討了其基因組電轉(zhuǎn)化宿主菌并獲得具有感染性的噬菌體顆粒的基本條件,同時(shí)通過PCR刪除技術(shù)去除基因組中的tRNA基因,并將改造后的噬菌體DNA再次轉(zhuǎn)入宿主菌體內(nèi),以期了解噬菌體tRNA基因的功能。最后雖然沒有獲得改造后的噬菌體顆粒,但為我們研究噬菌體基因功能以及對(duì)噬菌體進(jìn)行人工改造進(jìn)行了技術(shù)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,
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R378;R516
本文編號(hào)：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,
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R378;R516
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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2 黃建軍,胡曉梅,饒賢才,張克斌,金曉琳,周瑩冰,李明,申曉冬,朱軍民,胡福泉;銅綠假單胞菌噬菌體PaP2生物學(xué)特性的研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2004年13期
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