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LamB基因的克隆及其在肺炎克雷菌中的表达

时间:2019-04-19 21:15来源:毕业论文
以肺炎克雷伯氏菌株KPM1026的质粒DNA为模板,通过PCR技术扩增并克隆出Lam B膜孔蛋白基因,然后对PCR产物进行纯化,并将其连接到载体质粒pUCP24上,构建pUCP24∷Lam B重组质粒,测序结果完全正

摘要:本文主要是以肺炎克雷伯氏菌株KPM1026的质粒DNA为模板,通过PCR技术扩增并克隆出Lam B膜孔蛋白基因,然后对PCR产物进行纯化,并将其连接到载体质粒pUCP24上,构建pUCP24∷Lam B重组质粒,测序结果完全正确。将重组质粒pUCP24∷Lam B转化进入菌株DH5α,再将携带重组质粒的菌株DH5α涂布到含有5µg/ml庆大霉素的LB抗性平板上进行筛选。提取重组菌株的质粒并用HindIII、EcoRI这两种酶对其进行双酶切,然后经琼脂糖凝胶电泳分析,看到目的基因条带大小约为1.3kb,这表明目的基因已经成功转化进入菌株。再将重组质粒转化进入肺炎克雷伯氏菌菌株KPM1176和KPM2444中。用美罗培南、头孢他啶、氨曲南、左氢氟沙星这四种碳青霉烯类抗生素检测它们对携带重组质粒的菌株KPM1176、KPM2444的最低抑菌浓度(MIC),发现携带重组质粒pUCP24∷Lam B的肺炎克雷伯氏菌菌株KPM1176和KPM2444与携带有自连质粒pUCP24 的KPM1176和KPM2444它们在美罗培南、头孢他啶、氨曲南、左氢氟沙星这四个抗生素中的MIC值基本没什么变化。34668
毕业论文关键词:肺炎克雷伯氏菌,Lam B膜孔蛋白基因,碳青霉烯类药物,最低抑菌浓度(MIC)。
Cloning and expression of Lam B gene in Klebsiella pneumoniae
Abstract:This paper mainly using the genomic DNA of Klebsiella pneumoniae strain KPM1O26 as template,amplified and cloned membrane protein gene of Lam B using PCR technology, the PCR product was purified ,and connect it to the plasmid vector pUCP24, construct pUCP24: Lam B recombinant plasmid, sequencing results completely correct.The recombinant plasmid was transformed into B Lam pUCP24: Klebsiella pneumoniae strains KPM1176 and KPM2444, the resistance plate screening of recombinant strain of gentamicin 10mg/ml.The recombinant strain and plasmid with restriction endonuclease HindIII and EcoRI were digested, agarose gel electrophoresis analysis,fragment strip size is about 1.3kb, showed that the target gene was successfully transformed into strain and expression.Minimum inhibitory concentration of meropenem, ceftazidime, aztreonam, levofloxacin and the four carbapenems on recombinant Klebsiella pneumoniae strains KPM1176 and KPM2444 (MIC) were detected.The recombinant plasmid of Klebsiella pneumoniae strains KPM1176, KPM2444 and carry the empty vector of Klebsiella pneumoniae strains KPM1176, KPM2444 in meropenem, aztreonam, ceftazidime, levofloxacin four hydrogen left carbapenems in the same MIC value basically no change, namely Lam B film hole protein gene resistant to carbapenem resistance basically has no effect on Klebsiella pneumoniae strains. The Lam B membrane protein gene of resistance to carbapenem resistance basically has no effect on Klebsiella pneumoniae strains. 源¥自%六^^维*论-文+网=www.lwfree.cn
Keywords: Klebsiella pneumoniae, membrane protein gene Lam B, carbapenems, minimum inhibitory concentration (MIC).
目录
引言    1
1. 材料与方法    2
1.1供试材料    2
1.1.1菌株    2
1.1.2常用试剂和培养基的配制    2
1.1.3仪器设备    2
1.2实验方法    3
1.2.1 Lam B基因的克隆    3
1.2.2细菌的转化与筛选    5
1.2.3 菌株的MIC检测    7
2.结果与分析    7
2.1 Lam B基因克隆    8
2.2将重组质粒转化到DH5α    8
2.3 重组质粒转化进入菌株KPM1176和KPM2444的结果    9
2.4菌株的MIC结果    10
3. 讨论    11
致谢:    11
参考文献:    12
引言 肺炎克雷伯菌(KPN)是革兰氏阴性菌,它致病性较强,主要分布于人的呼吸道与肠道中[1]。它是感染菌和致病菌,常常会引发支气管炎,肺炎以及创伤部位的感染,有时甚至会导致败血症、脑膜炎和腹膜炎等疾病的发生。在最近十年里,由于青霉素以及头孢他啶类抗菌药物的大量生产使用,特别是第3代头孢他啶类抗菌药的投入使用,使得该菌对许多抗菌药物特别是对β-内酰胺类抗生素的耐药性处于不断提高中,而具有耐药性的菌株的出现使得人们对较严重的感染的治疗变得异常棘手[2-3]。它之所以对碳青霉烯类药物产生耐药性是因为:药物作用的靶位点出现了变化、细胞膜通透性出现了变化、产生能够破坏抗生素结构的酶、主动外排泵将进入细胞内的药物排出体外[4]。 LamB基因的克隆及其在肺炎克雷菌中的表达:http://www.lwfree.cn/shengwu/20190419/32257.html
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