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目的:产超广谱β-内酰胺酶是肠杆菌科细菌和肺炎克雷伯菌对β-内酰胺酶类抗生素耐药的重要机制,该类酶主要水解青霉素类、头孢菌素类以及单环酰胺类抗生素。自从1983年第一次有关β内酰胺酶的报道开始,至今已经发现超广谱β-内酰胺酶(ESBLs)的种类超过400种,其中SHV型是最常见的ESBLs类型之一。本研究主要探讨了超广谱β-内酰胺酶SHV-18的原核表达载体的构建、酶的纯化和活性验证。方法:利用肺炎克雷伯菌ATCC700603全基因组为模板,应用分子生物学技术钓取SHV-18基因,构建pET22b(+)-SHV-18表达质粒,经测序鉴定后,转化大肠杆菌Transetta(DE3),IPTG诱导表达并亲和纯化,进一步通过抗生素水解实验,检测其水解活性,测定其酶动力学参数。结果:成功构建了可以以裂解上清形式高效表达SHV-18的工程菌。通过亲和纯化最终获得纯度达到90%以上的β-内酰胺酶SHV-18。获得的SHV-18蛋白能够水解青霉素G、头孢拉定、头孢唑肟、头孢他啶、头孢吡肟等多个抗生素。结论:获得了对青霉素和头孢菌素类具有水解活性的超广谱β-内酰胺酶SHV-18,并对其酶动力学参数进行了检测。在实验室研究了β-内酰胺酶SHV-18对不同抗生素的水解特性,更加有利与指导临床抗生素的合理使用。
OBJECTIVE: To produce extended spectrum β-lactamases is an important mechanism of Enterobacteriaceae and Klebsiella pneumoniae resistance to β-lactamase antibiotics. These enzymes mainly hydrolyze penicillins, cephalosporins, Cyclic amide antibiotics. Since the first report on beta-lactamase in 1983, more than 400 ESBLs have been found, of which SHV is one of the most common types of ESBLs. In this study, we explored the construction of prokaryotic expression vector of extended-spectrum β-lactamase SHV-18, purification of enzyme and validation of its activity. Methods: The whole genome of Klebsiella pneumoniae ATCC700603 was used as a template. The SHV-18 gene was amplified by molecular biology techniques to construct the expression vector pET22b (+) - SHV-18. The recombinant plasmid was transformed into E.coli Transetta (DE3) , IPTG induced expression and affinity purification, further through the hydrolysis of antibiotics, hydrolysis activity was measured, and its kinetic parameters were measured. Results: The engineered bacteria that could efficiently express SHV-18 in the form of lysate supernatant were successfully constructed. By affinity purification, the purity of β-lactamase SHV-18 reached 90% or more. The obtained SHV-18 protein can hydrolyze penicillin G, cefradine, ceftizoxime, ceftazidime, cefepime and other antibiotics. Conclusion: The extended-spectrum β-lactamase SHV-18 with hydrolytic activity against penicillins and cephalosporins was obtained and its enzyme kinetic parameters were tested. In the laboratory study of β-lactamase SHV-18 hydrolysis of different antibiotics, more conducive and guide the rational use of clinical antibiotics.