【作者】 彭青；

【导师】 钱元恕；

【作者基本信息】 汕头大学 ， 药理学， 2005， 硕士

【摘要】 目的:对临床分离的嗜麦芽窄食单胞菌所产L1型金属β-内酰胺酶基因进行序列分析,明确酶基因表型;构建表达载体与嗜麦芽窄食单胞菌L1编码基因的重组体,并在大肠埃希菌BL21(DE3)中原核表达。检测L1的等电点,并测定L1金属β-内酰胺酶对多种β-内酰胺类抗生素的稳定性,以及L1对多种β内酰胺类抗生素的酶动力学参数,温度及pH等因素对酶活性的影响。 方法: (1)参照文献,设计合成L1的PCR引物,以临床分离的嗜麦芽窄食单胞菌710的基因组作为模板,PCR扩增L1酶的编码基因。将L1的PCR产物与pUCm—T载体连接,测定L1的核苷酸序列。 (2)将L1酶基因克隆至表达载体pET-41a(+),转化至宿主菌大肠埃希菌BL21(DE3)中。SDS-聚丙烯酰胺凝胶电泳检测表达情况。 (3)提取重组菌的β内酰胺酶粗提液,等电聚集电泳测定L1的等电点(pI)并观察酶抑制剂和锌离子对酶活性的影响。 (4)以分光光度计测定及比较多种β内酰胺类抗生素对L1型金属β-内酰胺酶及超广谱β内酰胺酶的酶稳定性,并测定L1酶对多种β内酰胺类抗生素的动力学参数,以及温度及pH等因素对酶活性的影响。 结果: (1)序列分析结果显示目的基因片断长873 bp,经GenBank同源性比较,目的基因的氨基酸序列与金属酶L1型编码基因blaS(注册更多还原

【Abstract】 Objective: To analyse the sequence of L1 metallo- β -lactamase encoding gene from clinical isolated stenotro-phomas maltophilia and construct the prokaryotic expression vector carrying L1 gene and expressed in E. coli BL21(DE3). To measure the pI recombinant L1 and test the stability and kinetic parametes of β -lactams hydrolysis of L1. To detect the activity of L1 in different pH and temperature.Methods:(1) The PCR primers of L1 gene were designed according to the literatures. The entire L1 encoding gene was amplified from the genome of clinical isolated stenotrophomonas maltophilia 710 by PCR and sequenced after ligated with pUCm-T vector.(2) L1 encoding gene was cloned into pET-41a(+) vector and the recombinant plasmid was transformed into E. coli BL21. Expression of L1 was comfirmed by SDS-PAGE electrophoresis.(3) Crude β -lactamase extracted from recombinant strain. The pi of recombinant L1 was determined by isoelectric focusing and influence of zink iron and inhibitor on L1 emzyme activity was observed.(4) The stability of L1 and ESBLs to different β -lactamswas compared and kinetic parametes were tested using spectr-ophotometer. The activity of LI emzyme in different pH and temperatures was observed by spectrotometer. Results:(1) The 873 bp DNA fragment of LI MBL encoding gene was amplified from strain 710 by PCR and aminoacid sequence analysis showed the target gene was 92.44% homologous to blaS of MBL LI.(2) After being inducted with IPTG, a 58 kDa recombinant fusion protein of GST and LI was expressed in the pET-41a(+) system.(3) Isoelectric focusing showed the pi of recombinant LI was 6. 7. The activity of emzyme can be inhibited by EDTA and relive by lOOMm ZnS04.(4) The recombinant LI’ s capacity of hydrolyzing penicillin G is similar to that of hydrolyzing cabapenem. Aztreonam and Ceftazidime are highly stable to LI. Ph 5. 8 and 40°C were the most suitable pH and temperature for LI. Substrate inhibition for LI emzyme appeared in high substrate concentration.更多还原