本文選題：霍亂弧菌 + 溶原性噬菌體��； 參考：《中國疾病預(yù)防控制中心》2016年博士論文

【摘要】：霍亂是由霍亂弧菌引起的急性腸道傳染病,據(jù)歷史記載已有七次霍亂全球大流行,目前仍未停息的第七次霍亂大流行,是由01群El Tor生物型引起。高守一等建立的“噬菌體-生物分型”方案,根據(jù)菌株的溶原性、對溶原噬菌體的敏感性、山梨醇發(fā)酵試驗(yàn)和溶血試驗(yàn)等4個生物學(xué)試驗(yàn),將El Tor型菌株分成12個(a-l)生物型,再結(jié)合噬菌體分型將埃爾托型菌株分為流行株和非流行株,為我國的霍亂防控做出了突出貢獻(xiàn)。對噬菌體-生物分型中的溶原性測定和對溶原性噬菌體的敏感性兩個分型指標(biāo)的機(jī)制進(jìn)行研究,有助于我們從表型差異入手,分析霍亂弧菌的分型機(jī)理、生物學(xué)變異和基因組變異。本研究首先對噬菌體-生物分型中對溶原性噬菌體的敏感性指標(biāo)中所用的溶原性噬菌體919TP進(jìn)行了全基因組測序和一般生物學(xué)特性分析。噬菌體919TP屬于肌尾噬菌體科,其最佳感染復(fù)數(shù)為1, 919TP感染宿主菌SM6的潛伏期約為60 min,爆發(fā)期約為60min,爆發(fā)量為4。噬菌體919P基因組為線性雙鏈DNA,長度為33,133bp,GC含量為48.92 %。經(jīng)過基因預(yù)測注釋及全基因組比對,確定了噬菌體919TP為K139家族噬菌體。接著我們對噬菌體919TP的抗性機(jī)制進(jìn)行了研究,選取01群El Tor型霍亂弧菌116株,檢測這些菌株對噬菌體919TP的敏感性,同時檢測這些菌株是否具有溶源化的919TP,菌株的LPS基因簇是否完整,最終發(fā)現(xiàn)在檢測菌株中,部分菌株LPS基因簇存在突變,但并不影響其對噬菌體919TP的敏感性;大多數(shù)菌株含有溶源化的919TP,利用超感染免疫對噬菌體919TP產(chǎn)生抗性,實(shí)驗(yàn)數(shù)據(jù)還證實(shí)這種免疫發(fā)生在噬菌體與細(xì)菌受體的結(jié)合之后;部分菌株由于LPS的突變從而導(dǎo)致噬菌體919TP與菌株不吸附,從而對919TP不感染;尚有部分菌株既不含有溶源化的919TP噬菌體基因組,又不含有突變的LPS序列,可能含有未知的抗噬菌體感染的機(jī)制而造成對噬菌體919TP的不感染,具體對噬菌體感染的機(jī)制尚需進(jìn)一步研究。再者我們對溶原性測定的機(jī)理進(jìn)行了研究,結(jié)果顯示01群El Tor型溶原性測定為陽性的菌株,其釋放的噬菌體主要為K139家族噬菌體,具有溶原化的K139噬菌體基因組,并對溶原性噬菌體919TP的敏感性為陰性;對溶原性噬菌體919TP的敏感性為陽性的01群El Tor型菌株,溶原性測定全部為陰性,基因組中不含有K139噬菌體;6株01群古典型菌株溶原性檢測為陽性,但產(chǎn)生的噬菌體不是K139類;0139群溶原性測定為陽性的菌株能檢測到溶原性K139噬菌體基因組,并且所有0139菌株都不能被919TP感染。最終確定了溶原性測定指標(biāo)主要檢測的是菌株釋放K139類噬菌體的能力,即是否有K139噬菌體的溶原化并釋放子代,“對溶原性噬菌體的敏感性”檢測的是菌株能否被K139家族噬菌體(919TP)感染,生物分型中“溶原性測定”與“對溶原性噬菌體的敏感性”兩個指標(biāo)是相關(guān)聯(lián)的。最后我們在霍亂弧菌古典株浙66菌株中發(fā)現(xiàn)并分離了一株新的溶原性噬菌體ZP66。通過對ZP66進(jìn)行基因組測定和一般特性分析,發(fā)現(xiàn)噬菌體ZP66屬于肌尾噬菌體科,基因組為線性雙鏈DNA,大小為33,499 bp, GC含量49.85 %,共預(yù)測到49個ORFs,覆蓋了94.78%的噬菌體全基因組序列。經(jīng)過基因預(yù)測注釋及蛋白序列比對,判定噬菌體ZP66為Mu類噬菌體。對噬菌體ZP66尾鞘蛋白基因進(jìn)行聚類分析顯示,噬菌體ZP66與霍亂弧菌中的同源基因聚為一個分支,并與其它弧菌和單胞菌聚為一個大的分支,這個聚類結(jié)果與細(xì)菌的分類相吻合。對噬菌體ZP66在霍亂弧菌中的宿主譜和流行情況進(jìn)行了分析,發(fā)現(xiàn)噬菌體ZP66只能感染O1群菌株,并且只在01群古典生物型中發(fā)現(xiàn)有ZP66基因組的存在,但只含有部分ZP66基因組的古典株,同樣能被噬菌體ZP66感染。通過對N16961、N16961-LPS缺失株和回補(bǔ)株對ZP66敏感性的檢測,推斷噬菌體ZP66的受體是01血清群的LPS。通過本研究,我們確定了噬菌體-生物分型中“溶原性測定”和“對溶原性噬菌體的敏感性”兩個指標(biāo)的分型機(jī)理,分析了霍亂弧菌天然分離株對噬菌體919TP的抗性機(jī)制,為我們了解噬菌體-生物分型機(jī)理提供了重要的理論基礎(chǔ),同時發(fā)現(xiàn)了一株新的溶原性噬菌體ZP66,為首次在霍亂弧菌分離到Mu類噬菌體。
[Abstract]:Cholera is an acute intestinal infectious disease caused by Vibrio cholerae. According to history, seven cholera global pandemics have been recorded, and the seventh cholera pandemic has not been stopped at present. It is caused by the 01 group of El Tor biotypes. The "phage biotyping" scheme established by Gao Shouyi, etc., is sensitive to the lylyphage phage sensitivity according to the lysin of the strain. 4 biological tests, such as sorbitol fermentation test and hemolysis test, divided the El Tor strain into 12 (A-L) biotypes, and then combined phage typing to divide the erto strains into epidemic and non epidemic strains, which made outstanding contributions to the prevention and control of cholera in China. The study of the mechanism of the two sensibility indexes helps us to analyze the genotyping mechanism, biological variation and genomic variation of Vibrio cholerae from the phenotypic difference. First, the whole genome sequencing of the lysoluble phage 919TP used in the sensitivity index of phage biotype to lytic phage was first sequenced. General biological characteristics analysis. Phage 919TP belongs to the myocutaneous phage family, the best infection complex number is 1, the incubation period of the 919TP infected host SM6 is about 60 min, the outbreak period is about 60min, the explosive amount of 4. phage 919P genome is linear double chain DNA, the length is 33133bp, and the GC content is 48.92%. The bacteriophage 919TP was identified as K139 family phage. Then the resistance mechanism of phage 919TP was studied. 01 groups of El Tor type Vibrio cholerae were selected and 116 strains of Vibrio cholerae were selected to detect the sensitivity of these strains to phage 919TP. At the same time, it was detected whether these strains had dissolved 919TP, and the LPS gene cluster of the strain was complete and finally found. Among the detected strains, some strains of LPS gene cluster have mutations, but they do not affect their sensitivity to phage 919TP; most of the strains contain soluble 919TP and use superinfection immunity to produce resistance to phage 919TP. The experimental data also confirm that the immunization occurs after the combination of phage with bacterial receptor; some strains are due to LPS. The mutation leads to no adsorption of phage 919TP and strain, which does not infect 919TP; some strains do not contain both the 919TP phage genome and the mutation LPS sequence, which may contain unknown anti phage infection mechanism and cause no infection to phage 919TP, and the mechanism of phage infection is still specific. Further study was needed. Furthermore, we studied the mechanism of LYSOGENICITY determination. The results showed that 01 groups of El Tor type LYSOGENICITY were positive, the phage released mainly was K139 family phage, the lysogenizing K139 phage genome, and the sensibility of the lysogen 919TP was negative, and the lysogen phage 91 was negative. The 01 group of El Tor strains with positive sensitivity were negative, and the lylygenicity was all negative. The genomes did not contain K139 phage; 6 strains of 01 groups of classical strains were positive, but the phage produced by the phage was not K139; 0139 groups of lylygenicity tested positive for the detection of the lytic K139 phage genome, and all 0139 The strains could not be infected by 919TP. Finally, the lyse determination index was determined mainly by the ability of the strain to release K139 phage, that is, whether there is the lylygenization of the K139 phage and release the progeny. "The sensitivity of the lytic phage" is that the strain can be infected by the K139 family phage (919TP), and the biotype "lytic" Sex determination "is associated with two indexes" sensitivity to lysogen phage. Finally, we found and separated a new lysogen phage ZP66. in the classical strain of Vibrio cholerae, Zhejiang 66, and found that the phage ZP66 belongs to the myocutaneous phage, and the genome is the genomes. Linear double stranded DNA, 33499 BP in size and 49.85% for GC, was predicted to 49 ORFs and covered 94.78% of the whole genome sequence of phage. The phage phage ZP66 was identified as Mu phage through gene prediction annotation and protein sequence alignment. The phage ZP66 tail sheath protein gene analysis showed that phage ZP66 and Vibrio cholerae were the same The source gene is clustered into one branch and is clustered with other Vibrio and monomonas sp. as a large branch. This clustering result is in accordance with the classification of bacteria. The host spectrum and epidemic situation of phage ZP66 in Vibrio cholerae are analyzed. It is found that phage ZP66 can only infect O1 group strains, and only found ZP66 in 01 groups of classical biologic forms. The existence of the genome, but the classical strain containing only part of the ZP66 genome, can also be infected by phage ZP66. By detecting the ZP66 sensitivity of N16961, N16961-LPS deletion and remedial plants, it is concluded that the receptor of phage ZP66 is the LPS. of the 01 serogroup, and we have determined the "lytic determination" in the phage biotyping and the "lytic determination" in the phage biotyping. The mechanism of two index typing for the sensitivity of lysogen phage was analyzed. The resistance mechanism of the natural isolates of Vibrio cholerae to phage 919TP was analyzed, which provided an important theoretical basis for us to understand the mechanism of phage biotyping, and a new lysogen phage ZP66 was discovered for the first time to isolate the Mu class from Vibrio cholerae. Phage.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 羅成;;Mu噬菌體的研究概況[J];微生物學(xué)雜志;1988年04期

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本文編號：1779255