Expression And Identification Of CTX-M-69 Gene And The Study On The Enzyme Characteristics Of The Gene Encoded Extended-spetrum Beta-lactamases

In the study,the complete gene of the CTX-M-1 type extended-spetrum beta-lactamases (ESBLs)was amplified by PCR,with the specific primers designed according to the CTX-M-1 type ESBLs sequence in Genbank,and the PCR template extracted from strains producing the CTX-M type ESBLs.Then the fragments were inserted into PGM-T vetor, and sequenced.The result of sequencing indicated that the length of the CTX-M type complete gene is 876 bp,encoding 291 aa.Confirmed by the internationalβ-lactamase authentication organization(http://www.lahey.org/Studies),the amplified sequence of this research is a new kind of CTX-M type ESBLs gene.Named as CTX-M-69 gene(GeneBank Access No.EU402393),protein series NO.ABY91281.1.Comparative analysis of CTX-M-69 gene and CTX-M-1 gene(GeneBank Access No.X92506) showed that they shared a nucleic acid homology at 99.0%(867/877),and an amino acid homology at 98.6%(287/291).The result of comparative analysis showed that CTX-M-69 gene belongs to CTX-M-1 cluster.Compared with CTX-M-1,There are ten nucleotide sites which occur mutation,six are nonsense mutation and four are sense mutation. Compared it with parts of other domestic and overseas CTX-M type gene,they shared a amino acid homoloty at more than98.9%..CTX-M-69 gene was amplified by PCR,with the template of CTX-M-69 gene clone vector as template,then inserted into pET-30b(+)to construct the expression plasmid of CTX-M-69 gene.The positive recombinate plasmid was transformed into E.coli BL21(DE3),and was induced by IPTG to express the ESBLs CTX-M-69.A fusion protein about 33kD was identified by SDS-PAGE.The expression product took the form of dissoluble protein and inclusion body.The optimal conditions to expree ESBLs CTX-M-69 were IPTG 0.4mmol/L,cultred at 30℃for 4h.In the optimal conditions,the content the recombinate ESBLs CTX-M-69 improved dramatically.The result of four antibiotic sensitivity tests(CTX,CAZ,CTX/CA,CAZ/CA)by K-B method showed it accord with the phenotype character of ESBLs,and the recombinate ESBLs CTX-M-69 strain produce ESBLs.The result of hydrolysis experiment of 12 beta-lactam antibiotics showed that the antibacterial spetrum was similar to CTX-M-1 type ESBLs except the hydrolysis of ceftazidime.The result showed that the activity of ESBLs CTX-M-69 was highest when the pH was 7.8～8.6,temperature of 30℃.The enzyme activity was restrained when the substrate concentration is too high.This research amplifed the complete gene of CTX-M type ESBLs by PCR,has discovered the new ESBLs CTX-M-69 gene,constructed the prokaryotic expression system of ESBLs CTX-M-69,achieved high efficient expression of ESBLs CTX-M-69, and has elementarily determined the kinetic parameter.The research has provided academic foundation for further research of ESBLs CTX-M-69 structure,biology activity and space conformation.