尿路致病性大肠埃希菌Ⅰ型菌毛fimH基因疫苗对小鼠的免疫保护性研究

发布时间：2018-06-07 11:48

  本文选题：尿路致病性大肠埃希菌 + Ⅰ型菌毛　； 参考：《山东大学》2011年硕士论文

【摘要】：目的：尿路感染(Urinary tract infection, UTI)是一种常见的临床疾病,引起UTI的致病菌约70%～95%为尿路致病性大肠埃希菌(Uropathogenic Escherichia coli, UPE)。UPEC的临床分离株对抗菌药物多表现为多重耐药,给其感染的治疗带来一定困难,故研制疫苗用以预防UPEC感染具有重要的现实意义。本实验构建尿路致病性大肠埃希菌Ⅰ型菌毛fimH基因真核表达载体,并研究尿路致病性大肠埃希菌Ⅰ型菌毛fimH基因核酸疫苗产生的免疫反应及对小鼠的免疫保护作用,同时对本疫苗经不同免疫途径产生的免疫效果进行初步比较。 方法： 1.提取尿路致病性大肠埃希菌临床株基因组DNA, PCR扩增fimH基因片段,然后克隆到TA载体,通过PCR、酶切及测序鉴定后,将f imH基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.0,构建pcDNA3.0-f imH重组质粒。通过PCR、酶切对重组质粒pcDNA3.0-f imH进行鉴定。 2.大量扩增并纯化质粒pcDNA3.0-f imH以免疫BABL/c小鼠。选取BALB/c小鼠40只,随机分为四组(PBS组、空质粒组、pcDNA3.0-fimH肌肉注射组、pcDNA3.0-fimH滴鼻组),使用本室构建的fimH基因的真核表达载体pcDNA3.0重组质粒分别通过肌肉注射和滴鼻(粘膜)2种途径免疫BALB/c小鼠,同时分别以载体质粒(pcDNA3.0)和PBS液为空质粒对照和空白对照,经股四头肌注射免疫小鼠,间隔两周,四组共连续免疫3次,检测小鼠血清中特异的IgG抗体。于末次免疫后第10天,以UPEC分离株菌液进行尿道上行攻击；攻击后第5天对小鼠及尿液进行细菌培养和计数(每只小鼠取尿液10μl接种于麦康凯平板。37℃培养24小时,第二天观察细菌菌落,进行菌落计数)。 结果： 1. pcDNA3.0-f imH重组质粒经PCR扩增获得了约910bp的片段；pcDNA3.0-fimH重组质粒经BamH I和XhoⅠ双酶切后,产生了1个与fimH基因PCR产物大小一致的小片段和1个不同于pcDNA3.0-f imH重组质粒的大片段,表明fimH基因已成功插入pcDNA3.0质粒中；测序获得的fimH DNA序列与GenBank登录的J 96株fimH DNA的编码序列基本一致。表明成功构建了尿路致病性大肠埃希菌fimH基因真核表达载体pcDNA3.0-fimH;。 2.将质粒免疫小鼠可诱导小鼠产生抗Ⅰ型菌毛抗体。免疫小鼠后,肌注组与滴鼻组小鼠血清特异性[gG抗体均明显升高,高于对照组及空质粒组(P0.05)； 3. UPEC分离株菌液攻击小鼠后,进行小鼠尿液菌落计数,pcDNA3.0-fimH肌注组与滴鼻组小鼠尿液菌落计数明显少于对照组及空质粒组(P0.05)。 结论： 1.成功构建尿路致病性大肠埃希菌Ⅰ型菌毛fimH基因真核表达质粒pcDNA3. O-fimH; 2.动物实验结果表明,pcDNA3.0-fimH作为基因疫苗,可诱导BALB/c小鼠产生特异的体液免疫,对小鼠尿道上行感染具有一定的免疫保护作用；不同的免疫途径免疫效果亦有不同。
[Abstract]:Objective: urinary tract infection (Uropathogenic tract infection, UTI) is a common clinical disease. About 70% of the pathogens causing UTI are Uropathogenic Escherichia coli. The clinical isolates of UPE).UPEC are multidrug resistant to antimicrobial agents. It is difficult to treat UPEC infection, so it is very important to develop vaccine to prevent UPEC infection. In this study, the eukaryotic expression vector of fimH gene of pathogenic Escherichia coli type I pili was constructed, and the immune response of fimH gene nucleic acid vaccine of pathogenic Escherichia coli type I to mice was studied. At the same time, the immune effect of the vaccine produced by different immune routes was compared preliminarily. Methods: 1. The genomic DNAs of pathogenic Escherichia coli were extracted, the fimH gene fragment was amplified by PCR, then cloned into TA vector. The f imH gene fragment was digested by restriction endonuclease after identification by PCR, enzyme digestion and sequencing. PcDNA3.0-f imH recombinant plasmid was constructed by cloning into eukaryotic expression vector pcDNA3.0. The recombinant plasmid pcDNA3.0-f imH was identified by restriction endonuclease digestion. 2. Plasmid pcDNA3.0-f imH was amplified and purified to immunize BABL/c mice. Forty BALB/c mice were randomly divided into four groups. The empty plasmid group (pcDNA3.0-fimH intramuscular injection group) was used to immunize BALB/c mice by intramuscular injection and nasal drip. The eukaryotic expression vector pcDNA3.0 recombinant plasmid of fimH gene was injected intramuscularly and intramuscularly. At the same time, the vector plasmid pcDNA3.0) and the PBS solution were used as empty plasmid control and blank control respectively. The mice were immunized with quadriceps femoris muscle respectively. The mice were immunized continuously for three times with the interval of two weeks, to detect the specific IgG antibody in the serum of the mice. On the 10th day after the last immunization, urethral uplink attack was carried out with the UPEC isolate, and the mice and urine were cultured and counted on the 5th day after the attack (10 渭 l of urine was taken from each mouse and cultured at Kang Kai plate. 37 鈩，

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