本文選題：釀酒酵母 + 多藥耐藥轉(zhuǎn)運蛋白��； 參考：《浙江大學》2012年碩士論文

【摘要】：微生物耐藥是抗菌治療中面臨的嚴峻挑戰(zhàn)之一。近年來,致病真菌的多藥物耐藥現(xiàn)象日益增多,導致免疫缺陷、器官移植等病人抗感染治療難度增大,治療費用昂貴,臨床致死率較高。導致多藥物耐藥現(xiàn)象的一個重要原因是細胞中ABC轉(zhuǎn)運蛋白過量表達。Pdr5p是釀酒酵母中重要的ABC轉(zhuǎn)運蛋白,它與許多致病酵母菌株中發(fā)現(xiàn)的ABC轉(zhuǎn)運蛋白在結(jié)構(gòu)和功能上高度相似。以Pdr5p為模型,研究ABC轉(zhuǎn)運蛋白介導的多藥物耐藥性分子機制已成為近年的研究熱點。 本論文采用PCR介導的隨機突變方法,對高表達質(zhì)粒YEplac195：BPDR5進行突變,在氟康唑藥物板上篩選到兩個能導致Pdr5p外排泵功能嚴重受損的單一位點突變C793F和S1230F。其中C793位于預測的Pdr5p跨膜螺旋6(TMH6)的胞內(nèi)邊界上,而S1230位于跨膜螺旋7(TMH7)的胞外邊界處。通過研究兩個突變株的耐藥性,發(fā)現(xiàn)其在含有放線菌酮、羅丹明6G、紅四氮唑的固體藥物板上具有和PDR5缺失菌株相同的耐藥表型。在含有上述藥物不同濃度的液體培養(yǎng)基中,通過測定相對生長率,發(fā)現(xiàn)C793F和S1230F突變株對藥物的外排能力也顯著下降。Pdr5p特異性ATP酶活性實驗和Western blot實驗表明793和1230的突變沒有導致Pdr5p特異性ATP酶活性改變,突變的Pdr5p依然能夠正確定位在細胞膜上且蛋白表達量基本沒有受到影響,說明這兩個位點突變造成Pdr5p功能受損的原因是氨基酸的差異性。 為了進一步研究Pdr5p功能受損的機制,我們通過定點突變分別構(gòu)建了C793S, C793M,C793Y, S1230A,S1230N, S1230Y等單一位點系列突變株。通過研究這些突變菌株的藥物敏感性、Pdr5p特異性ATP酶活以及蛋白定位、表達等,發(fā)現(xiàn)所有突變菌株均具有和WT菌株相似的的ATP酶活性和膜定位,但其藥物敏感性則與替代的氨基酸的性質(zhì)相關(guān)。實驗的結(jié)果表明在793位點,氨基酸空間體積改變是導致Pdr5p功能受損的主要因素；在1230位點,氨基酸的空間體積和極性的變化共同影響Pdr5p的外排泵功能。 本論文在隨機突變的基礎上篩選出能導致Pdr5p功能嚴重受損的兩個新突變位點,通過藥物篩選和生化證據(jù)證實位于跨膜區(qū)兩端的位點對于Pdr5p的功能至關(guān)重要。這些結(jié)果為進一步明晰ABC轉(zhuǎn)運蛋白的分子機制提供了新的證據(jù)。
[Abstract]:Microbial resistance is one of the severe challenges in antimicrobial therapy.In recent years, the phenomenon of multidrug resistance of pathogenic fungi is increasing day by day, which leads to more difficult anti-infection treatment of patients such as immune deficiency, organ transplantation, high cost of treatment and high clinical mortality.One of the important reasons for multidrug resistance is that the overexpression of ABC transporter. Pdr5p is an important ABC transporter in Saccharomyces cerevisiae. It is highly similar to the ABC transporter found in many pathogenic yeast strains in structure and function.Using Pdr5p as a model to study the molecular mechanism of multidrug resistance mediated by ABC transporter has become a hot topic in recent years.In this paper, PCR mediated random mutation was used to mutate the high expression plasmid YEplac195:BPDR5. Two single site mutations, C793F and S1230F, were screened on fluconazole drug plate, which could seriously damage the function of Pdr5p efflux pump.C793 is located at the intracellular boundary of the predicted Pdr5p transmembrane helix 6 (TMH6), while S1230 is located at the extracellular boundary of the transmembrane helix 7 (TMH7).By studying the drug resistance of two mutants, it was found that they had the same drug resistance phenotype as PDR5 deficient strains on the solid drug plate containing actinomycin, Rhodamine 6G and erythrotetrazole.In liquid media containing different concentrations of the above drugs, the relative growth rate was measured.It was found that the efflux ability of C793F and S1230F mutants also decreased significantly. Pdr5p-specific ATP enzyme activity test and Western blot assay showed that the mutation of 793 and 1230 did not result in the change of Pdr5p specific ATP enzyme activity.The mutated Pdr5p can still be located correctly on the cell membrane and the protein expression is not affected, which indicates that the difference of amino acid is the reason of the damage of Pdr5p function caused by the mutation of these two loci.In order to further study the mechanism of Pdr5p function impairment, we constructed C793S, C793MN C793Y, S1230Agna S1230N, S1230Y single locus series by site-directed mutagenesis (S793S), C793Y, S1230Y and S1230Y, respectively.By studying the drug-sensitive Pdr5p specific ATP enzyme activity and protein localization and expression of these mutant strains, it was found that all mutant strains had similar ATP enzyme activity and membrane localization to WT strain.However, its drug sensitivity is related to the properties of the substituted amino acids.The results showed that at site 793, the change of amino acid spatial volume was the main factor leading to the impairment of Pdr5p function, and at site 1230, the spatial volume and polarity of amino acid affected the function of Pdr5p efflux pump.On the basis of random mutation, two new mutation sites were screened out which can seriously damage the function of Pdr5p. The results of drug screening and biochemical evidence confirm that the sites located at the two ends of the transmembrane region are very important for the function of Pdr5p.These results provide new evidence for further understanding the molecular mechanism of ABC transporter.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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