Studies on the function of the encoding gene of TEM-105 type of ?-lactamases / 中国药理学通报

ABSTRACT

Aim To obtain the encoding gene sequences of TEM-type ?-lactamases produced by 4-strain Klebsiella pneumoniae in Zhejian g Province, identify their genotypes and study some properties of these TEM-typ e ?-lactamases.Methods The encoding genes of TEM-type ?-la ctamases produced by 4 isolates were amplified by PCR. The purified PCR products were ligated with pGEM-T easy vectors, expressed in E. coli DH 5?, and sequenced by Sanger's dideoxy chain termin ation composition method. Compared with anino acid sequences in the GenBank,TEM -types of the ?-lactamases was determined. The genes of TEM ?-lactamases were ligated with pET-28 c vector to express recombinant proteins in E. coli DH 5?. Plasmids were extracted from the p ronucleus expression strains and PCR was performed to determine whether the pron ucleus expression was successful or not. Their phenotypes were determined by ESB Ls phenotype affirmative test. The isoelectric points (pIs) of the recombinant p roteins were determined by isoelectric focus. Conjugation test was performed to determine whether their genes existed in plasmid or chromosome. Results The encoding genes of ?-lactamases were determined as TEM by PCR. It s PCR product had 1 009 nucleotides. The pI of the novel TEM ?-lactamase was 5.4. The enzyme was determined as non-ESBLs by ESBLs phenotype affirmative tes t.Transconjugants were successfully selected from the paternal producers in conj ugation tests. The TEM-type ?-lactamase produced by 4 strains was determined as TEM-105(AF516720) by GenBank. Conclusion The ?-lactamase produced by 4-strain K. pneumoniae from 4 patients in Zhejiang Province was TEM-105. It was the first report of TEM-105 type ?-lactamase produced by 4-st ain K. pneumoniae from China in the world.