鸡源大肠杆菌TEM-57型β-内酰胺酶基因的原核表达及酶特性

Prokaryotic expression of TEM-57 type β-lactamase gene produced by fowl Escherichia coli and its related characteristics

对该临床菌株进行接合试验,采用PCR扩增获得blaTEM-57基因片段,定向克隆入表达载体pET-28a构建重组质粒,经酶切和DNA测序鉴定后进行诱导表达,用双紫外分光光度仪测定表达蛋白的底物水解特性和抑制剂的抑酶特性。结果显示,耐药质粒成功从供体菌接合转移到大肠杆菌C600,接合子对青霉素类及氨基糖苷类药物耐药。PCR扩增产物电泳出现blaTEM-57目的条带。重组质粒经EcoRⅠ、XholⅠ双酶切及测序鉴定后,显示pET-28α/TEM-57原核表达载体构建成功。原核表达的最优条件为IPTG浓度0.8mmol/L、温度37℃、诱导时间4h。blaTEM-57能水解氨苄西林、阿莫西林、头孢氨苄、头孢噻呋钠、头孢曲松钠、头孢噻肟钠及头孢哌酮,对头孢氨苄的Km最小为2.4,对头孢噻呋钠的Km最大为121.5;抑制剂舒巴坦钠和他唑巴坦对blaTEM-57水解抗生素有不同程度的抑制作用。pET-28α/TEM-57重组质粒得到成功构建和诱导表达,为进一步研究酶的其他特性及制备特异性的单克隆抗体奠定了基础。

Objective of this study was to carry out the prokaryotic expression of blaTEM-57 type β-lactamase gene produced by fowl Escherichia coli,and identify related enzyme characteristics of the expressed products.Conjugation test was performed to determine whether the gene existed,the amplification product by PCR was subcloned into expression vector pET-28α to constructe the recombinant plasmid.Identified via dige-stion by restriction enzymes and DNA sequencing,blaTEM-57 type β-lactamase was induced to express.The characteristics of antibiotic hydrolysis and inhibition of β-lactamase inhibitors on blaTEM-57 type β-lactamase were observed with ultraviolet spectrophotometer.Results revealed that the positive plasmid were transmitted into recipient C600 successfully,transconjugants were resistant to penicillins and aminoglycosides.The blaTEM-57 gene was cloned and expressed in prokaryote cell successfully.The optimized conditions of expressing recombinant plasmid strains were 0.8 mmol/L IPTG,and 37℃ induced temperatures,and 4h induced times,respectively.The blaTEM-57 can hydrolyze ampicillin,amoxicillin,cefalexin,ceftiofur,ceftriaxone,cefotaxime and cepoperazon,Km value to cefalexin was 2.4,to ceftiofur was 121.5.Sulbactam and tazobactam could effectively inhibited hydrolysis of blaTEM-57 on antibiotics.Recombinate plasmids of pET-28α/TEM-57 have been constructed and expressed successfully,which laid the foundation for further study on its other characteristics and preparation of specific monoclonal antibody.