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LamB基因的克隆及其在肺炎克雷菌中的表達

時間:2019-04-19 21:15來源:畢業論文
以肺炎克雷伯氏菌株KPM1026的質粒DNA為模板,通過PCR技術擴增并克隆出Lam B膜孔蛋白基因,然后對PCR產物進行純化,并將其連接到載體質粒pUCP24上,構建pUCP24∷Lam B重組質粒,測序結果完全正

摘要:本文主要是以肺炎克雷伯氏菌株KPM1026的質粒DNA為模板,通過PCR技術擴增并克隆出Lam B膜孔蛋白基因,然后對PCR產物進行純化,并將其連接到載體質粒pUCP24上,構建pUCP24∷Lam B重組質粒,測序結果完全正確。將重組質粒pUCP24∷Lam B轉化進入菌株DH5α,再將攜帶重組質粒的菌株DH5α涂布到含有5µg/ml慶大霉素的LB抗性平板上進行篩選。提取重組菌株的質粒并用HindIII、EcoRI這兩種酶對其進行雙酶切,然后經瓊脂糖凝膠電泳分析,看到目的基因條帶大小約為1.3kb,這表明目的基因已經成功轉化進入菌株。再將重組質粒轉化進入肺炎克雷伯氏菌菌株KPM1176和KPM2444中。用美羅培南、頭孢他啶、氨曲南、左氫氟沙星這四種碳青霉烯類抗生素檢測它們對攜帶重組質粒的菌株KPM1176、KPM2444的最低抑菌濃度(MIC),發現攜帶重組質粒pUCP24∷Lam B的肺炎克雷伯氏菌菌株KPM1176和KPM2444與攜帶有自連質粒pUCP24 的KPM1176和KPM2444它們在美羅培南、頭孢他啶、氨曲南、左氫氟沙星這四個抗生素中的MIC值基本沒什么變化。34668
畢業論文關鍵詞:肺炎克雷伯氏菌,Lam B膜孔蛋白基因,碳青霉烯類藥物,最低抑菌濃度(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^^維*論-文+網=www.aftnzs.live
Keywords: Klebsiella pneumoniae, membrane protein gene Lam B, carbapenems, minimum inhibitory concentration (MIC).
目錄
引言    1
1. 材料與方法    2
1.1供試材料    2
1.1.1菌株    2
1.1.2常用試劑和培養基的配制    2
1.1.3儀器設備    2
1.2實驗方法    3
1.2.1 Lam B基因的克隆    3
1.2.2細菌的轉化與篩選    5
1.2.3 菌株的MIC檢測    7
2.結果與分析    7
2.1 Lam B基因克隆    8
2.2將重組質粒轉化到DH5α    8
2.3 重組質粒轉化進入菌株KPM1176和KPM2444的結果    9
2.4菌株的MIC結果    10
3. 討論    11
致謝:    11
參考文獻:    12
引言 肺炎克雷伯菌(KPN)是革蘭氏陰性菌,它致病性較強,主要分布于人的呼吸道與腸道中[1]。它是感染菌和致病菌,常常會引發支氣管炎,肺炎以及創傷部位的感染,有時甚至會導致敗血癥、腦膜炎和腹膜炎等疾病的發生。在最近十年里,由于青霉素以及頭孢他啶類抗菌藥物的大量生產使用,特別是第3代頭孢他啶類抗菌藥的投入使用,使得該菌對許多抗菌藥物特別是對β-內酰胺類抗生素的耐藥性處于不斷提高中,而具有耐藥性的菌株的出現使得人們對較嚴重的感染的治療變得異常棘手[2-3]。它之所以對碳青霉烯類藥物產生耐藥性是因為:藥物作用的靶位點出現了變化、細胞膜通透性出現了變化、產生能夠破壞抗生素結構的酶、主動外排泵將進入細胞內的藥物排出體外[4]。 LamB基因的克隆及其在肺炎克雷菌中的表達:http://www.aftnzs.live/shengwu/20190419/32257.html
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