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

【摘要】：近年來,CTX-M型超廣譜β-內酰胺酶(ESBLs)的出現(xiàn)給臨床治療帶來了極大的挑戰(zhàn),三種CTX-M雜合酶(CTX-M-64、-123和-132)已經在我國動物源大腸桿菌中檢測到,它們對大多數頭孢菌素類表現(xiàn)出增強的水解活性,其基因序列暗示了這些雜合基因可能是通過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力學參數,結合蛋白空間結構分析其催化作用機制,并探索雜合基因的可能形成途徑,為CTX-M酶的進一步研究提供科學依據及參考數據。本研究通過PCR方法擴增出雜合酶(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)及不含信號肽的序列,并將其插入p ET-28b表達載體。重組質粒轉入E.coli BL21(DE3),分別用于MIC值的測定及蛋白表達。在蛋白表達水平一致的情況下,測定攜帶blaCTX-Ms全長的六株重組菌對各種β-內酰胺類抗生素的MIC值。結果顯示,產CTX-M-14的菌株對除了氨芐西林外的所有受試抗生素的MIC值最低,產CTX-M-64、CTX-M-132、CTX-M-123和CTX-M-55的菌株對頭孢噻肟和頭孢他啶的MIC值高于CTX-M-15,其中CTX-M-64對頭孢噻肟的MIC值最高。整體來看,六種blaCTX-Ms基因的耐藥性水平從低到高的順序為: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-內酰胺類抑制劑(克拉維酸、他唑巴坦和舒巴坦)對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,與頭孢噻肟聯(lián)用后的MIC比單獨使用頭孢噻肟時減小高達近70000倍。SDS-PAGE結果顯示,六個目的蛋白的大小均約為28k Da,最佳表達條件是IPTG1.0mmol/L,30℃誘導培養(yǎng)5h。通過親和層析和凝膠過濾層析方法得到純度大于99%的目的蛋白,用于酶動力學參數的測定。結果顯示,這些酶的催化活性(kcat/Km)與MIC結果相似,CTX-M-14對除了頭孢噻吩外的所有β-內酰胺類抗生素的催化活性最低,而CTX-M-64對頭孢硝基噻吩、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對除了氨芐西林外的β-內酰胺類抗生素的催化活性均低于CTX-M-55及三個雜合酶CTX-M-64、-132和-123的催化活性。另外,三種抑制劑對六種CTX-Ms酶的抑制水平均在納摩級,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。結合酶動力學參數,對六種CTX-Ms酶的氨基酸序列及空間結構進行比較分析。結果發(fā)現(xiàn),CTX-M-55與CTX-M-15相差僅一個氨基酸殘基A77V,但CTX-M-55對超廣譜頭孢菌素類藥物表現(xiàn)出相對較強的催化活性。結構分析顯示,第77位氨基酸位于活性位點遠端,CTX-M-55更可能是通過V77與不同α螺旋上的關鍵氨基酸形成疏水鍵,從而穩(wěn)定蛋白空間結構中螺旋群的核心架構并促成更加穩(wěn)定的活性部位構象。穩(wěn)定構象的形成促成了CTX-M-55的較高結構穩(wěn)定性,進而表現(xiàn)出了較高的催化效率和對溫度變化的耐受性。從CTX-M-15、CTX-M-132、CTX-M-123到CTX-M-64的演變是通過向CTX-M-15中逐漸引入活性中心遠端殘基的過程,并且在演變過程中它們對超廣譜頭孢菌素類藥物的催化活性也在逐漸增強。與CTX-M-14相比,CTX-M-64對頭孢菌素類增強的催化活性及對β-內酰胺酶抑制劑增強的敏感性大部分也是由于活性中心遠端殘基的引入。這些結果表明,活性中心遠端的氨基酸殘基增強了CTX-M酶對超廣譜頭孢菌素類藥物的催化活性。通過PCR測序的方法,對實驗室保存的分離自2010~2013年的大腸桿菌檢測blaCTX-M雜合體基因,并對陽性大腸桿菌進行多位點序列分型。通過接合轉移或化學轉化的方法獲得攜帶雜合酶基因和其可能來源基因bla_(CTX-M-15)和bla_(CTX-M-55)單一質粒的接合子或轉化子,S1-PFGE鑒定質粒大小,并對質粒進行復制子分型。對分別攜帶bla_(CTX-M-15)、bla_(CTX-M-64)、bla_(CTX-M-123)和bla_(CTX-M-132)的四個質粒p HNY2-1、p HNAH46-1、p HNAH4-1和p HNLDH19進行高通量測序并分析其結構。結果發(fā)現(xiàn),攜帶bla_(CTX-M-15)的Inc I2質粒p HNY2-1、攜帶bla_(CTX-M-55)的質粒p HN1122-1、攜帶bla_(CTX-M-64)的質粒p HNAH46-1和攜帶bla_(CTX-M-132)的質粒p HNLDH19都含有相同的插入序列ISEcp1-blaCTX-M-Inc A/C,并且質粒骨架幾乎一致,說明雜合基因可能起源于bla_(CTX-M-15)。攜帶bla_(CTX-M-123)的質粒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blaCTX-M-Inc A/C-Inc I2轉移到Inc I1型質粒上。采用PCR測序及限制性內切酶片段長度多態(tài)性分析(RFLP)等方法進一步研究p HNAH46-1和p HNAH4-1的相似質粒在動物源大腸桿菌中的擴散情況。結果從不同省份、不同農場和不同動物中分別檢測出5株攜帶有與p HNAH46-1相似的Inc I2質粒和9株攜帶有與p HNAH4-1相似的Inc I1(ST108)質粒,這表明攜帶雜合基因bla_(CTX-M-64)和bla_(CTX-M-123)的質粒已經在動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.
【學位授予單位】：華南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q55;Q78

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