超广谱β-内酰胺酶SHV-18的表达纯化及活性验证

The Expression,Purification and Biological Activity of Extended-Spectrum β-Lactamase(ESBLs) SHV-18

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： The production of ESBLs through plasmid is the mainly mechanism of Enterobacteriaceae and Klebsiella pneumoniae to resist β-1actamase antibiotics, ESBLs are able to hydrolyse penicillins, eephalosporin and single cyclic arnide. Since the first report about β-1actamase in 1983, researchers from all over the world have found more than 400 kinds of ESBLs, among them, SHV is one of the most common ones. This research mainly discuss the construction of prokaryotic expression vector of SHV-18, the purification and biological activity of the enzyme. Methods： Using microbiological technology, we obtain gene SHV-18 from E.coli ATCC35218 and Cray Borrelia bacteria pneumonia ATCC700603, and then we constructed the expression plasmid pET22b（＋）-SHV-18. The expression vector was confirmed by sequecing and transformed into Transetta （DE3）, induced by IPTG to express the recombinant SHV-18 and purification the protein. Then detect hydrolyse activity by hydrolysis antibiotics. Results： We construct SHV-18 engineering bacteria existe protein in the supematant of lysis successfully, after protein expression and purification, the purity of the target protein is higher than 90%. And the protein can hydrolyse various antibiotics ,such as PenicillinG, Cefradine, Ceftizoxime, Ceflazidime, Cefepime and so on. Conclusion： We obtain the extended-spectrum beta-lactamase SHV-18 which is able to hydrolyse penicillins, cephalosporin, and we detect its dynamical parameter. We have a research on the characteristics of SHV- 18 hydrolyse various antibiotics. It will contribute to the reasonable use of clinical antibiotics.