本文選題：超廣譜β-內(nèi)酰胺酶 + CTX-M雜合體��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：近年來(lái),CTX-M型超廣譜β-內(nèi)酰胺酶(ESBLs)的出現(xiàn)給臨床治療帶來(lái)了極大的挑戰(zhàn),三種CTX-M雜合酶(CTX-M-64、-123和-132)已經(jīng)在我國(guó)動(dòng)物源大腸桿菌中檢測(cè)到,它們對(duì)大多數(shù)頭孢菌素類(lèi)表現(xiàn)出增強(qiáng)的水解活性,其基因序列暗示了這些雜合基因可能是通過(guò)bla_(CTX-M-14)和blaCTX-M-15的重組形成。本文旨在比較雜合酶及其母體酶的動(dòng)力學(xué)參數(shù),結(jié)合蛋白空間結(jié)構(gòu)分析其催化作用機(jī)制,并探索雜合基因的可能形成途徑,為CTX-M酶的進(jìn)一步研究提供科學(xué)依據(jù)及參考數(shù)據(jù)。本研究通過(guò)PCR方法擴(kuò)增出雜合酶(CTX-M-132、-123和-64)及其母體酶(CTX-M-55、-15和-14)的編碼基因完整序列(876bp)及不含信號(hào)肽的序列,并將其插入p ET-28b表達(dá)載體。重組質(zhì)粒轉(zhuǎn)入E.coli BL21(DE3),分別用于MIC值的測(cè)定及蛋白表達(dá)。在蛋白表達(dá)水平一致的情況下,測(cè)定攜帶blaCTX-Ms全長(zhǎng)的六株重組菌對(duì)各種β-內(nèi)酰胺類(lèi)抗生素的MIC值。結(jié)果顯示,產(chǎn)CTX-M-14的菌株對(duì)除了氨芐西林外的所有受試抗生素的MIC值最低,產(chǎn)CTX-M-64、CTX-M-132、CTX-M-123和CTX-M-55的菌株對(duì)頭孢噻肟和頭孢他啶的MIC值高于CTX-M-15,其中CTX-M-64對(duì)頭孢噻肟的MIC值最高。整體來(lái)看,六種blaCTX-Ms基因的耐藥性水平從低到高的順序?yàn)?bla_(CTX-M-14)、bla_(CTX-M-15)、bla_(CTX-M-55)、bla_(CTX-M-132)、bla_(CTX-M-123)和bla_(CTX-M-64)。β-內(nèi)酰胺類(lèi)抑制劑(克拉維酸、他唑巴坦和舒巴坦)對(duì)六種酶都有較好的抑制作用,其中克拉維酸和他唑巴坦的抑制作用較強(qiáng),與頭孢噻肟聯(lián)用后的MIC比單獨(dú)使用頭孢噻肟時(shí)減小高達(dá)近70000倍。SDS-PAGE結(jié)果顯示,六個(gè)目的蛋白的大小均約為28k Da,最佳表達(dá)條件是IPTG1.0mmol/L,30℃誘導(dǎo)培養(yǎng)5h。通過(guò)親和層析和凝膠過(guò)濾層析方法得到純度大于99%的目的蛋白,用于酶動(dòng)力學(xué)參數(shù)的測(cè)定。結(jié)果顯示,這些酶的催化活性(kcat/Km)與MIC結(jié)果相似,CTX-M-14對(duì)除了頭孢噻吩外的所有β-內(nèi)酰胺類(lèi)抗生素的催化活性最低,而CTX-M-64對(duì)頭孢硝基噻吩、頭孢呋辛、頭孢噻呋、頭孢曲松和頭孢噻肟的催化活性最高。CTX-M-15對(duì)除了氨芐西林外的β-內(nèi)酰胺類(lèi)抗生素的催化活性均低于CTX-M-55及三個(gè)雜合酶CTX-M-64、-132和-123的催化活性。另外,三種抑制劑對(duì)六種CTX-Ms酶的抑制水平均在納摩級(jí),其中舒巴坦的IC50和Ki值高于克拉維酸,而克拉維酸略高于他唑巴坦。結(jié)合酶動(dòng)力學(xué)參數(shù),對(duì)六種CTX-Ms酶的氨基酸序列及空間結(jié)構(gòu)進(jìn)行比較分析。結(jié)果發(fā)現(xiàn),CTX-M-55與CTX-M-15相差僅一個(gè)氨基酸殘基A77V,但CTX-M-55對(duì)超廣譜頭孢菌素類(lèi)藥物表現(xiàn)出相對(duì)較強(qiáng)的催化活性。結(jié)構(gòu)分析顯示,第77位氨基酸位于活性位點(diǎn)遠(yuǎn)端,CTX-M-55更可能是通過(guò)V77與不同α螺旋上的關(guān)鍵氨基酸形成疏水鍵,從而穩(wěn)定蛋白空間結(jié)構(gòu)中螺旋群的核心架構(gòu)并促成更加穩(wěn)定的活性部位構(gòu)象。穩(wěn)定構(gòu)象的形成促成了CTX-M-55的較高結(jié)構(gòu)穩(wěn)定性,進(jìn)而表現(xiàn)出了較高的催化效率和對(duì)溫度變化的耐受性。從CTX-M-15、CTX-M-132、CTX-M-123到CTX-M-64的演變是通過(guò)向CTX-M-15中逐漸引入活性中心遠(yuǎn)端殘基的過(guò)程,并且在演變過(guò)程中它們對(duì)超廣譜頭孢菌素類(lèi)藥物的催化活性也在逐漸增強(qiáng)。與CTX-M-14相比,CTX-M-64對(duì)頭孢菌素類(lèi)增強(qiáng)的催化活性及對(duì)β-內(nèi)酰胺酶抑制劑增強(qiáng)的敏感性大部分也是由于活性中心遠(yuǎn)端殘基的引入。這些結(jié)果表明,活性中心遠(yuǎn)端的氨基酸殘基增強(qiáng)了CTX-M酶對(duì)超廣譜頭孢菌素類(lèi)藥物的催化活性。通過(guò)PCR測(cè)序的方法,對(duì)實(shí)驗(yàn)室保存的分離自2010~2013年的大腸桿菌檢測(cè)blaCTX-M雜合體基因,并對(duì)陽(yáng)性大腸桿菌進(jìn)行多位點(diǎn)序列分型。通過(guò)接合轉(zhuǎn)移或化學(xué)轉(zhuǎn)化的方法獲得攜帶雜合酶基因和其可能來(lái)源基因bla_(CTX-M-15)和bla_(CTX-M-55)單一質(zhì)粒的接合子或轉(zhuǎn)化子,S1-PFGE鑒定質(zhì)粒大小,并對(duì)質(zhì)粒進(jìn)行復(fù)制子分型。對(duì)分別攜帶bla_(CTX-M-15)、bla_(CTX-M-64)、bla_(CTX-M-123)和bla_(CTX-M-132)的四個(gè)質(zhì)粒p HNY2-1、p HNAH46-1、p HNAH4-1和p HNLDH19進(jìn)行高通量測(cè)序并分析其結(jié)構(gòu)。結(jié)果發(fā)現(xiàn),攜帶bla_(CTX-M-15)的Inc I2質(zhì)粒p HNY2-1、攜帶bla_(CTX-M-55)的質(zhì)粒p HN1122-1、攜帶bla_(CTX-M-64)的質(zhì)粒p HNAH46-1和攜帶bla_(CTX-M-132)的質(zhì)粒p HNLDH19都含有相同的插入序列ISEcp1-blaCTX-M-Inc A/C,并且質(zhì)粒骨架幾乎一致,說(shuō)明雜合基因可能起源于bla_(CTX-M-15)。攜帶bla_(CTX-M-123)的質(zhì)粒p HNAH4-1為Inc I1型(ST108),含有ISEcp1-bla_(CTX-M)-Inc A/C-Inc I2片段,提示ISEcp1介導(dǎo)blaCTX-M-Inc A/C-Inc I2轉(zhuǎn)移到Inc I1型質(zhì)粒上。采用PCR測(cè)序及限制性?xún)?nèi)切酶片段長(zhǎng)度多態(tài)性分析(RFLP)等方法進(jìn)一步研究p HNAH46-1和p HNAH4-1的相似質(zhì)粒在動(dòng)物源大腸桿菌中的擴(kuò)散情況。結(jié)果從不同省份、不同農(nóng)場(chǎng)和不同動(dòng)物中分別檢測(cè)出5株攜帶有與p HNAH46-1相似的Inc I2質(zhì)粒和9株攜帶有與p HNAH4-1相似的Inc I1(ST108)質(zhì)粒,這表明攜帶雜合基因bla_(CTX-M-64)和bla_(CTX-M-123)的質(zhì)粒已經(jīng)在動(dòng)物源大腸桿菌中廣泛傳播。
[Abstract]:In recent years, the emergence of CTX-M type broad-spectrum beta lactamase (ESBLs) has brought great challenges to clinical treatment. Three kinds of CTX-M heterozygases (CTX-M-64, -123 and -132) have been detected in our animal source Escherichia coli, which exhibit enhanced hydrolysis activity for most cephalosporins. The gene sequences suggest that these heterozygous genes are available. It can be formed by the recombination of bla_ (CTX-M-14) and blaCTX-M-15. This paper aims to compare the kinetic parameters of heterozygase and its maternal enzyme, analyze its catalytic mechanism by combining the spatial structure of protein, and explore the possible formation pathway of heterozygous gene, and provide scientific basis and reference data for further research of CTX-M enzyme. This study through the PCR method The complete sequence of the encoding gene (876bp) and its maternal enzyme (CTX-M-55, -15 and -14) and its maternal enzyme (CTX-M-55, -15 and -14) were amplified and inserted into the P ET-28b expression vector. The recombinant plasmid was transferred into E.coli BL21 (DE3) and used to determine the value and the protein expression respectively. In the case of the same protein expression level, the recombinant plasmid was used to determine the protein expression. The MIC value of various beta lactam antibiotics was carried by six recombinant strains of blaCTX-Ms full length. The results showed that the MIC value of all the tested antibiotics except ampicillin was the lowest, and the MIC value of the strains producing CTX-M-64, CTX-M-132, CTX-M-123 and CTX-M-55 to cefotaxime and ceftazidime was higher than CTX-M-15, including CTX-M-64. The MIC value of cefotaxime is the highest. Overall, the order of the resistance of the six blaCTX-Ms genes from low to high is bla_ (CTX-M-14), bla_ (CTX-M-15), bla_ (CTX-M-55), bla_ (CTX-M-132), bla_ (CTX-M-123) and the inhibitor of beta lactam (clavulanic acid, tazobactam and sulbactam), which have a better inhibition on the six enzymes. The inhibitory effect of clavulanic acid and tazobactam was stronger. The MIC of cefotaxime compared with cefotaxime was nearly 70000 times higher than that of cefotaxime. The results showed that the size of six target proteins was about 28K Da, the best expression was IPTG1.0mmol/L, and 5h. was induced by affinity chromatography and gel filtration at 30 C. The target protein with purity greater than 99% was obtained by chromatography and used for the determination of enzyme kinetic parameters. The results showed that the catalytic activity of these enzymes (kcat/Km) was similar to that of MIC, and CTX-M-14 had the lowest catalytic activity for all beta lactam antibiotics except cephalothiophene, while CTX-M-64 was the cephalosporin, cefuroxime, ceftif, head, and ceftathiophene. The catalytic activity of cefotaxime and cefotaxime was the highest.CTX-M-15 activity of the beta lactam antibiotics except ampicillin was lower than that of CTX-M-55 and three heterozygous enzymes CTX-M-64, -132 and -123. In addition, the inhibition levels of the three inhibitors on the six CTX-Ms enzymes were at the namo level, and the IC50 and Ki values of the sulbactam were higher than those of the sulbactam. Clavulanic acid, while clavulanic acid was slightly higher than tazobactam. The amino acid sequence and spatial structure of the six CTX-Ms enzymes were compared. The results showed that the difference between CTX-M-55 and CTX-M-15 was only one amino acid residue A77V, but CTX-M-55 showed a relatively strong catalytic activity for the hyper broad-spectrum cephalosporin. The analysis shows that the seventy-seventh bit amino acid is located at the far end of the active site, and CTX-M-55 is more likely to form a key by V77 and the key amino acids on the different alpha helix, thus stabilizing the core structure of the helix group in the spatial structure of the protein and facilitating a more stable conformation of the active site. The formation of stable texture contributes to the higher structural stability of CTX-M-55. The evolution of CTX-M-15, CTX-M-132, CTX-M-123 to CTX-M-64 is through the gradual introduction of the distal residues of the active center to CTX-M-15, and their catalytic activity to the hyper broad-spectrum cephalosporin is gradually enhanced during the evolution. And CTX-M Compared with -14, the catalytic activity of the cephalosporins enhanced by CTX-M-64 and the sensitivity to the enhancement of beta lactamase inhibitors are mostly due to the introduction of the distal residues of the active center. These results suggest that the amino acid residues at the distal end of the active center enhance the catalytic activity of the CTX-M enzyme to the Hyper broad-spectrum cephalosporin. By PCR sequencing Methods the blaCTX-M heterozygous gene was detected from the Escherichia coli isolated from 2010~2013 in the laboratory, and the multi point sequence of the positive Escherichia coli was typed. The zygote of the heterozygous gene and the possible source gene bla_ (CTX-M-15) and the bla_ (CTX-M-55) single plasmid were obtained by conjugation transfer or chemical transformation. The plasmid size was identified by S1-PFGE, and the plasmids were typed. The four plasmid P HNY2-1, which carried bla_ (CTX-M-15), bla_ (CTX-M-64), bla_ (CTX-M-123) and bla_ (CTX-M-132), were sequenced and analyzed by high flux sequencing. 1, plasmid P HN1122-1 carrying bla_ (CTX-M-55), bla_ (CTX-M-64) plasmid P HNAH46-1 and plasmid P HNLDH19 carrying bla_ (CTX-M-132) all contain the same insertion sequence, and the plasmid skeleton is almost identical, indicating that the heterozygous gene may be derived from the plasmid. The NC I1 type (ST108) contains ISEcp1-bla_ (CTX-M) -Inc A/C-Inc I2 fragment, suggesting that ISEcp1 mediated blaCTX-M-Inc A/C-Inc I2 is transferred to the plasmids. Results 5 strains of Inc I2 plasmids similar to P HNAH46-1 were detected in different provinces, different farms and different animals, and 9 strains of Inc I1 (ST108) plasmids similar to P HNAH4-1 were carried out. This showed that the plasmid carrying heterozygous gene bla_ (CTX-M-64) and bla_ were widely spread in E. coli from animal sources.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q55;Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前4條

1 苑麗;劉建華;胡功政;潘玉善;莫娟;魏永俊;裴亞玲;;雞大腸桿菌TEM和CTX-M型超廣譜β-內(nèi)酰胺酶基因分型研究[J];中國(guó)農(nóng)業(yè)科學(xué);2010年20期

2 陳劍;李君文;邱志剛;郭聰;諶志強(qiáng);汲珊珊;陳照立;王新為;金敏;;CTX-M型產(chǎn)ESBLs耐藥基因在城市典型河流中的生態(tài)分布[J];應(yīng)用與環(huán)境生物學(xué)報(bào);2014年01期

3 杜向黨;焦顯芹;莫娟;張素梅;侯春彬;李新生;;雞豬源大腸桿菌CTX-M型ESBLs的分子檢測(cè)[J];華北農(nóng)學(xué)報(bào);2009年02期

4 ;[J];;年期

相關(guān)會(huì)議論文 前5條

1 呂霞;李從榮;李艷;;湖北地區(qū)產(chǎn)CTX-M型超廣譜β內(nèi)酰胺酶肺炎克雷伯菌的分子流行病學(xué)研究[A];第五次全國(guó)中青年檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2006年

2 劉文恩;;湖南地區(qū)CTX-M型超廣譜β-內(nèi)酰胺酶的研究[A];第五次全國(guó)中青年檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2006年

3 肖代雯;楊永長(zhǎng);喻華;劉華;黃文芳;廖維金;胡琦;黃波;;產(chǎn)CTX-M型超廣譜β-內(nèi)酰胺酶大腸埃希菌基因型分析[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議資料匯編[C];2008年

4 趙曉麗;胡大春;邵劍春;蔣杰;周玲;劉德華;;產(chǎn)ESBLs大腸埃希菌CTX-M型耐藥基因分析[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議資料匯編[C];2008年

5 李向陽(yáng);方曄;楊錦紅;;亞抑菌濃度抗生素對(duì)攜帶CTX-M型基因質(zhì)粒接合轉(zhuǎn)移的影響[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議資料匯編[C];2008年

相關(guān)博士學(xué)位論文 前1條

1 賀丹丹;CTX-M型雜合酶生化特性及遺傳特征研究[D];華南農(nóng)業(yè)大學(xué);2016年

相關(guān)碩士學(xué)位論文 前2條

1 趙曉麗;大腸埃希菌CTX-M型ESBLs研究[D];昆明醫(yī)學(xué)院;2007年

2 馮建昆;鴨源大腸桿菌CTX-M型ESBLs的基因亞型檢測(cè)及基因環(huán)境研究[D];河南農(nóng)業(yè)大學(xué);2013年

，

本文編號(hào)：1991129