本文關(guān)鍵詞： 阪崎克羅諾桿菌 抑制性消減雜交 基因敲除 毒力相關(guān)基因　出處：《天津科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：阪崎克羅諾桿菌(Cronobater sakazakii)是一種條件致病菌,能在人和動物的腸道內(nèi)寄生的一種無芽孢的革蘭氏陰性桿菌。這一致病菌能導(dǎo)致各個年齡段的人感染,對新生兒的威脅較大,能引起新生兒腦膜炎,菌血癥和壞死性小腸結(jié)腸炎,死亡率達40%-80%。此外,還能引起成人的局部感染、骨髓炎和菌血癥等。目前的研究顯示阪崎克羅諾桿菌的毒力具有多樣性,乳鼠灌胃實驗表明不同阪崎克羅諾桿菌菌株之間的毒力存在明顯差異。雖然阪崎克羅諾桿菌已經(jīng)成為國內(nèi)外研究的焦點,但是其致病機制仍然不夠清楚。 為了獲得阪崎克羅諾桿菌的毒力相關(guān)基因,本研究利用腹腔注射ICR小鼠對24株食源性分離株的毒力進行了檢測,根據(jù)致死時間判斷不同菌株的毒力大小。從中選擇毒力最高和毒力最低的菌株,利用抑制性雜交(SSH)篩選其差異基因,結(jié)果共獲得7個差異片段,測序后將結(jié)果輸入NCBI數(shù)據(jù)庫進行BlastN與BlastX比對分析,確定基因性質(zhì)。其中基因pap(編碼菌毛亞基轉(zhuǎn)運與菌毛組裝相關(guān)蛋白,可能與阪崎克羅諾桿菌的致病性有關(guān) 為了進一步確認(rèn)pap(基因的毒力相關(guān)性,利用基因敲除同源重組技術(shù)構(gòu)建了pap(基因缺失突變株ΔpapC,通過PCR技術(shù)對缺失突變株進行了確認(rèn)。將108CFU野生株與突變株ΔpapC分別腹腔注射出生三天的SD乳鼠,記錄乳鼠死亡時間,結(jié)果顯示野生株的平均致死時間為6.5小時,而突變株的平均致死時間為9.625小時,ΔpapC突變株毒力明顯降低。將野生株與缺失突變株作梯度稀釋,分別腹腔注射SD乳鼠,記錄不同注射劑量實驗組動物的死亡情況,計算半致死劑量(LD50),結(jié)果顯示野生株的LDso為3.162×106CFU,突變株的LD50為8.511×106CFU,突變株的半致死劑量提高了2.69倍。以上動物實驗證實了papC基因與菌株的毒性相關(guān)。本研究為阪崎克羅諾桿菌致病機制的研究奠定了基礎(chǔ)。
[Abstract]:Cronobater sakazakii), a conditional pathogen that parasitizes in the intestines of humans and animals, is a spore-free gram-negative bacillus that causes infections in people of all ages and poses a greater threat to newborns. It can cause neonatal meningitis, bacteremia and necrotizing enterocolitis, with a mortality rate of 40 to 80. In addition, it can cause local infection, osteomyelitis and bacteremia in adults. The virulence of different strains of Crosenia sakazakii was obviously different from that of lactation. Although the virulence of Hanshazaki Crosenia has become the focus of research at home and abroad, the pathogenetic mechanism of the virulence is still unclear. In order to obtain virulence related genes of Hanshizaki Crosenia, the virulence of 24 foodborne isolates was detected by intraperitoneal injection of ICR mice. According to the lethal time, the virulence of different strains was judged. The most virulent and least virulent strains were selected, and the differentially expressed genes were screened by inhibition hybridization SSH. After sequencing, the results were input into the NCBI database for BlastN and BlastX comparison analysis to determine the gene properties. The gene PAPs (encoding bacterial fimbrial subunit transport and fimbriae assembly related proteins) may be related to the pathogenicity of Hanshizaki Crotonia. To further confirm the virulence of the gene, The deletion mutant 螖 PAPCwas constructed by gene knockout homologous recombination technique, and the deletion mutant was confirmed by PCR technique. The 108CFU wild strain and mutant strain 螖 papC were injected intraperitoneally into SD newborn rat after three days of birth, and the time of death was recorded. The results showed that the average lethal time of wild strain was 6.5 hours, while that of mutant strain was 9.625 hours. The virulence of 螖 papC mutant was significantly decreased. To record the death of animals in the experimental group with different injection doses, The results showed that the LDso of wild strain was 3.162 脳 10 ~ (6) CFU, the LD50 of mutant was 8.511 脳 10 ~ (6) CFU, and the half lethal dose of mutant was increased by 2.69 times. More animal experiments confirmed that the papC gene was related to the toxicity of the strain. The study of pathogenic mechanism of Noreobacter spp.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378.1;Q75

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