阪崎克罗诺杆菌毒力相关基因的筛选鉴定

发布时间：2018-02-21 11:09

本文关键词： 阪崎克罗诺杆菌抑制性消减杂交基因敲除毒力相关基因　出处：《天津科技大学》2012年硕士论文　论文类型：学位论文

【摘要】：阪崎克罗诺杆菌(Cronobater sakazakii)是一种条件致病菌,能在人和动物的肠道内寄生的一种无芽孢的革兰氏阴性杆菌。这一致病菌能导致各个年龄段的人感染,对新生儿的威胁较大,能引起新生儿脑膜炎,菌血症和坏死性小肠结肠炎,死亡率达40%-80%。此外,还能引起成人的局部感染、骨髓炎和菌血症等。目前的研究显示阪崎克罗诺杆菌的毒力具有多样性,乳鼠灌胃实验表明不同阪崎克罗诺杆菌菌株之间的毒力存在明显差异。虽然阪崎克罗诺杆菌已经成为国内外研究的焦点,但是其致病机制仍然不够清楚。为了获得阪崎克罗诺杆菌的毒力相关基因,本研究利用腹腔注射ICR小鼠对24株食源性分离株的毒力进行了检测,根据致死时间判断不同菌株的毒力大小。从中选择毒力最高和毒力最低的菌株,利用抑制性杂交(SSH)筛选其差异基因,结果共获得7个差异片段,测序后将结果输入NCBI数据库进行BlastN与BlastX比对分析,确定基因性质。其中基因pap(编码菌毛亚基转运与菌毛组装相关蛋白,可能与阪崎克罗诺杆菌的致病性有关为了进一步确认pap(基因的毒力相关性,利用基因敲除同源重组技术构建了pap(基因缺失突变株ΔpapC,通过PCR技术对缺失突变株进行了确认。将108CFU野生株与突变株ΔpapC分别腹腔注射出生三天的SD乳鼠,记录乳鼠死亡时间,结果显示野生株的平均致死时间为6.5小时,而突变株的平均致死时间为9.625小时,ΔpapC突变株毒力明显降低。将野生株与缺失突变株作梯度稀释,分别腹腔注射SD乳鼠,记录不同注射剂量实验组动物的死亡情况,计算半致死剂量(LD50),结果显示野生株的LDso为3.162×106CFU,突变株的LD50为8.511×106CFU,突变株的半致死剂量提高了2.69倍。以上动物实验证实了papC基因与菌株的毒性相关。本研究为阪崎克罗诺杆菌致病机制的研究奠定了基础。
[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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.1;Q75

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