本文選題：尿路致病性大腸埃希菌 + Ⅰ型菌毛　； 參考：《山東大學(xué)》2011年碩士論文

【摘要】：目的：尿路感染(Urinary tract infection, UTI)是一種常見的臨床疾病,引起UTI的致病菌約70%～95%為尿路致病性大腸埃希菌(Uropathogenic Escherichia coli, UPE)。UPEC的臨床分離株對抗菌藥物多表現(xiàn)為多重耐藥,給其感染的治療帶來一定困難,故研制疫苗用以預(yù)防UPEC感染具有重要的現(xiàn)實(shí)意義。本實(shí)驗(yàn)構(gòu)建尿路致病性大腸埃希菌Ⅰ型菌毛fimH基因真核表達(dá)載體,并研究尿路致病性大腸埃希菌Ⅰ型菌毛fimH基因核酸疫苗產(chǎn)生的免疫反應(yīng)及對小鼠的免疫保護(hù)作用,同時(shí)對本疫苗經(jīng)不同免疫途徑產(chǎn)生的免疫效果進(jìn)行初步比較。 方法： 1.提取尿路致病性大腸埃希菌臨床株基因組DNA, PCR擴(kuò)增fimH基因片段,然后克隆到TA載體,通過PCR、酶切及測序鑒定后,將f imH基因片段用限制性內(nèi)切酶切下,克隆至真核表達(dá)載體pcDNA3.0,構(gòu)建pcDNA3.0-f imH重組質(zhì)粒。通過PCR、酶切對重組質(zhì)粒pcDNA3.0-f imH進(jìn)行鑒定。 2.大量擴(kuò)增并純化質(zhì)粒pcDNA3.0-f imH以免疫BABL/c小鼠。選取BALB/c小鼠40只,隨機(jī)分為四組(PBS組、空質(zhì)粒組、pcDNA3.0-fimH肌肉注射組、pcDNA3.0-fimH滴鼻組),使用本室構(gòu)建的fimH基因的真核表達(dá)載體pcDNA3.0重組質(zhì)粒分別通過肌肉注射和滴鼻(粘膜)2種途徑免疫BALB/c小鼠,同時(shí)分別以載體質(zhì)粒(pcDNA3.0)和PBS液為空質(zhì)粒對照和空白對照,經(jīng)股四頭肌注射免疫小鼠,間隔兩周,四組共連續(xù)免疫3次,檢測小鼠血清中特異的IgG抗體。于末次免疫后第10天,以UPEC分離株菌液進(jìn)行尿道上行攻擊；攻擊后第5天對小鼠及尿液進(jìn)行細(xì)菌培養(yǎng)和計(jì)數(shù)(每只小鼠取尿液10μl接種于麥康凱平板。37℃培養(yǎng)24小時(shí),第二天觀察細(xì)菌菌落,進(jìn)行菌落計(jì)數(shù))。 結(jié)果： 1. pcDNA3.0-f imH重組質(zhì)粒經(jīng)PCR擴(kuò)增獲得了約910bp的片段；pcDNA3.0-fimH重組質(zhì)粒經(jīng)BamH I和XhoⅠ雙酶切后,產(chǎn)生了1個(gè)與fimH基因PCR產(chǎn)物大小一致的小片段和1個(gè)不同于pcDNA3.0-f imH重組質(zhì)粒的大片段,表明fimH基因已成功插入pcDNA3.0質(zhì)粒中；測序獲得的fimH DNA序列與GenBank登錄的J 96株fimH DNA的編碼序列基本一致。表明成功構(gòu)建了尿路致病性大腸埃希菌fimH基因真核表達(dá)載體pcDNA3.0-fimH;。 2.將質(zhì)粒免疫小鼠可誘導(dǎo)小鼠產(chǎn)生抗Ⅰ型菌毛抗體。免疫小鼠后,肌注組與滴鼻組小鼠血清特異性[gG抗體均明顯升高,高于對照組及空質(zhì)粒組(P0.05)； 3. UPEC分離株菌液攻擊小鼠后,進(jìn)行小鼠尿液菌落計(jì)數(shù),pcDNA3.0-fimH肌注組與滴鼻組小鼠尿液菌落計(jì)數(shù)明顯少于對照組及空質(zhì)粒組(P0.05)。 結(jié)論： 1.成功構(gòu)建尿路致病性大腸埃希菌Ⅰ型菌毛fimH基因真核表達(dá)質(zhì)粒pcDNA3. O-fimH; 2.動(dòng)物實(shí)驗(yàn)結(jié)果表明,pcDNA3.0-fimH作為基因疫苗,可誘導(dǎo)BALB/c小鼠產(chǎn)生特異的體液免疫,對小鼠尿道上行感染具有一定的免疫保護(hù)作用；不同的免疫途徑免疫效果亦有不同。
[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，

本文編號(hào)：1991067