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目的检测产NDM-1肺炎克雷伯菌CS309是否同时携带产IMP、VIM型金属β-内酰胺酶或KPC型碳青霉烯酶的耐药基因,同时构建NDM-1基因原核表达质粒,并在大肠埃希菌中进行表达。方法采用聚合酶链反应(PCR)扩增IMP、VIM和KPC耐药基因;以产NDM-1肺炎克雷伯菌CS309为DNA模板,PCR扩增NDM-1全长,并将其与pGEM-T克隆载体连接后转化至大肠埃希菌DH5α,继而对阳性克隆进行双酶切,将酶切片段与pET-28α(+)表达载体连接,并转化大肠埃希菌BL21,再用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,并采用SDS-PAGE和Western blot技术验证NDM-1蛋白。结果经PCR和测序证实,该菌同时携带NDM-1和IMP-4两种金属酶基因,未扩增出VIM、KPC耐药基因。经双酶切和测序证实,原核表达质粒pET-28α(+)-NDM-1构建成功。SDS-PAGE发现,重组菌株经诱导后在28 kDa附近有明显条带,与预期蛋白大小27.9 kDa一致。Western blot表明诱导产生的融合蛋白可与NDM-1抗体特异性结合。结论肺炎克雷伯菌CS309同时携带NDM-1和IMP-4两种金属酶基因;成功构建了NDM-1基因的原核表达质粒,该质粒在大肠埃希菌BL21中高效融合表达。
Objective To detect whether the NDM-1-producing Klebsiella pneumoniae CS309 harboring the resistance gene of IMP, VIM-type metallo-β-lactamase or KPC-type carbapenema and construct the prokaryotic expression plasmid of NDM-1 gene simultaneously Expression in Escherichia coli. Methods Polymerase chain reaction (PCR) was used to amplify the resistance genes of IMP, VIM and KPC. The full-length NDM-1 was amplified by PCR with Klebsiella pneumoniae CS309 producing NDM-1 and then ligated with pGEM- T cloning vector was transformed into Escherichia coli DH5α, then the positive clones were double-digested, the digested fragment was ligated with pET-28α (+) expression vector and transformed into Escherichia coli BL21, then isopropyl β-D-thiogalactoside (IPTG) induced protein expression, and NDM-1 protein was verified by SDS-PAGE and Western blot. Results PCR and sequencing confirmed that the strain carried both NDM-1 and IMP-4 metalloenzyme genes and did not amplify VIM and KPC resistance genes. Double digestion and sequencing confirmed that prokaryotic expression plasmid pET-28α (+) - NDM-1 was successfully constructed. SDS-PAGE showed that the recombinant strain showed obvious bands around 28 kDa after induction, which was consistent with the expected protein size of 27.9 kDa. Western blot showed that the induced fusion protein could specifically bind to NDM-1 antibody. Conclusions The Klebsiella pneumoniae CS309 carries both NDM-1 and IMP-4 metalloenzyme genes. The prokaryotic expression plasmid of NDM-1 gene was successfully constructed and expressed in E. coli BL21 efficiently.