Enzymological Study Of VIM-2 Metallo-β-lactamase

1. Objective:To analyse the resistance actuality of Pseudomonas aeruginosa to imipenem and the sequence of VIM-2 metallo-β-lactamase encoding gene from clinical isolated Pseudomonas aeruginosa; to construct the prokaryotic expression vector carrying VIM-2 gene and expressed in E.coli BL21(DE3); to measure pI of the fusion protein and test the stability and kinetic parametes of β-lactams hydrolysis of VIM-2 and detect the activity of VIM-2 in different pH and temperature.2. Methods:2. 1 PCR amplificatios were performed with universal bla_IMP,bla_VIM,bla_SPM-1 and bla_OprD2 primers,and the IMP, VIM, SPM-1 and OprD₂ encoding gene were amplified from the genome of 46Pseudomonas aeruginosa.2. 2 The PCR primers of VIM-2 gene were designed according to the literatures. The entireVIM-2 encoding gene was amplified from the genome of clinical isolated Pseudomonasaeruginosa by PCR and sequenced after ligated with pUCm-T vector.2. 3 VIM-2 encoding gene was cloned into pET-41b(+) vector and the recombinant plasmid wastransformed into E.coli BL21. Expression and purification of VIM-2 was comfirmed bySDS-PAGE electrophoresis.2. 4 pI of the purified protein was determined by isoelectric focusing electrophoresis.2. 5 The stability of kinetic parametes of VIM-2 to different β-lactams and the activity of VIM-2 in different pH and temperature were tested using spectrophotometer.3. Results:3. 1 There was no Pseudomonas aeruginosa with encoding gene of the acquired metallo- β -lactamase and 7 with encoding gene of OprD₂.3. 2 The 801 bp DNA fragment of VIM-2 encoding gene was amplified from the strain Pseudomonas aeruginosa by PCR and amino acid sequence analysis showed the target gene was 100% homologous to the other VIM-2.