论文标题:禽源大肠杆菌链霉素耐药质粒的分离与鉴定及其耐药基因的流行病学研究
Isolating and Identification of Plasmid Coding Streptomycin Resistance from the Avian Escherichia Coli and Epidemiology of the Drug Resistant Genes
论文作者
论文导师 秦爱建,论文学位 硕士,论文专业 预防兽医学
论文单位 扬州大学,点击次数 69,论文页数 46页File Size888K
2006-05-01论文网 http://www.lw23.com/lunwen_265442192/
E coli; PCR; Streptomycin resistance; Toinactive enzyme
本研究通过19种常用药物的药敏实验,发现本实验室分离保存的216株禽源大肠杆菌对链霉素的耐药率为75%。提取部分链霉素高度耐药菌株的质粒,发现菌株AE6、AA7、AE9、BA4、BE、AE2具有相似的质粒图谱且均含有一大小在23Kb大小质粒。其中BE菌株除对磷霉素和利福平敏感外,对其余药物全部耐药。且BE菌株只携带有2种质粒,除23Kb大小质粒外,另一种质粒小于2Kb。将BE株这两种质粒DNA转化DH-5α,链霉素药物平板筛选获得一链霉素抗性转化株。提取转化株质粒,以BE株的质粒溶液作参照跑电泳,发现BE株的大质粒成功转入此转化株中。转化株的药敏实验发现此质粒确实编码了链霉素的耐药特性,这些结果证明23kb的质粒与细菌链霉素耐药基因有关。 根据GenBank发表的大肠杆菌链霉素耐药基因aadA1、strA及strB的序列分别设计引物,应用PCR实验技术对实验室保存的216株禽源大肠杆菌三种链霉素耐药基因进行检测,结果表明:aadA1、strA和strB的阳性率分别为49.1%,56%和65.7%。这些结果与药敏实验结果基本成正相关,但进一步比较发现,部分具有链霉素耐药基因的菌株对链霉素有敏感性,且检出率为51.8%。将相应基因测序,分析发现,AD5和AC3两菌株所携带strB耐药基因在45位点增加了一段序列GTTTCGC,在突变位点后产生了多个终止密码子,导致strB基因不能编码链霉素磷酸转移酶,证明插入突变导致了耐药菌株转变为敏感株。
I have gained one plasmids which coding streptomycin resistance through the convert experiment. Drug sensitivity tests were done to verify the transformant gained Streptmycin resistance .and PCR experiment shows that this R plasmid phore aadA1 gene onjy. Sequencing result finds that sequence of the aadA1 gene in the Rplasmid shares more than 98.2% homology with the gene aadA1 signed in GenBank .The data come up to 100% when it is translated into aa sequence. Gene coding streptomycin resistance such as aadA1、strA、strB were amplified by pojymerase chain reaction (PCR) from Avian Escherichia coli isolated from sick poultry. The primers were designed according to the sequence of aadA1、StrA、StrB gene published in GenBank . It intrests me that some strains which contain the Sr gene are sensitive to streptomycin.I suppose it is because the Sr gene have been mutanted or they exist as one intron in the chromatosome. Probabjy these strains will be turned into ones resistant to the Streptmycin not long after. Sequent result and homology anajyses prove that these three kinds of Sr gene share more than 98% homology with the sequence cited from GenBank. The percentage of aadA1 gene is 98.2%,pet of strA is 99.8%,strB is 99.9%.Wha’s noteworthy is that the strB gene in the AC3 and AD5 strain are both inserted with one piece of sequence (GTTTCGC). .Most inportant of all, there exists more than one terminator following the inserted sequence. It is possibly one new reason why the Sr gene strB can not encode toinactive enzyme.
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