The Molecular Biotechnology And Its Related Characterization Of The Extend Spectrum β-lactamase Produced By The Shigella Isolated From The Fowl

In this test,a standard shigella flcxncri was firstly induced by Ceftifur in order to making it producing ESBLs.Secondly the genotypes of ESBLs produced by the induced strain and five shigella flexneri clinical isolates were detected,respectly. The molecular envolvement mechanism of the shigella flexneri producing ESBLs was discussed by the intercomparsion of sub-genotypes.After that,the ESBLs sub-genotypes were expressed and the main facts affecting the expression levels were optiumed.At last,the experiments of ESBLs hydrolyzingβ-lactam antibotics and inhabited byβ-lactamase inhibitors were conducted to decide the hydrolytic ratios of enzymes against different substracts and the protective ratios ofβ-lactamase inhibitors against enzymes.The conlusion of genotypes detection:there were the same TEM-type and SHVtype sequences in the plasmids of the five shigeUa flexneri clinical isolates.The sequence of theTEM-type was characterized by two nucleotide mutations (G157A,C409T)compared with the sequence of AY903309(TEM-116).The mutation of nucleotide 409 was aslient mutation,the mutation of nucleotide 157 leaded to the mutation of amino acid(Gly53Ser)and the nucleotide sequence was the same as that priviously detected from a shigella flexneri clinical isolate in our laborary,the nucleotide sequence accession number is DY 211973.The nucleotide sequence of SHV-type was the same as that of the AY826418(SHV-12),The nucleotide sequence was deposited in the EMBL/GenBank databases through DDBJ and assigned accession number EF125011.For the standard shigella flexneri,when it was induced to the tenth times by Ceftiofur,its gene sequence of the producedβ-lactamase was the TEM-1 type,when induced to the fifth times,the gene sequence of the produced ESBLs was the TEM-1V type.Otherwise,the standard strain also harbors TEM type homoerotic nucleotide sequence.Sequence analysis revealed that the sequence of TEM-1 differed from the standard strain harboring TEM type homoerotic nucleotide sequence by one mutations(A787G)that led to aminoacid substitutions:Thr263Ala. Four point mutations(G157A,A250G,C409T,T551C)were found in the coding region of the blaTEM-1V gene compared with the blaTEM-1 gene,and these mutations caused three amino acid substitutions,Gly53Ser,Ile84Val,Val184Ala.The sequences of the blaTEM-1 genes have been given GenBank accession numbersEF125012.The conclusion of ESBLs genotypes:The blaSHV-12 genes and blaTEM-1V genes were expressed in E.coli BL21(DE3)through PET-28a vector,respectly.The expressed proteins own activation.The optimum expression condation of S-BL21 and T-BL21 are:S-BL21,IPTG concenration 0.5mmol/L,the optimum temperature 37℃,the optimum time 4h;T-BL21,IPTG concenration 0.8mmol/L,the optimum temperature 37℃,the optimum time 4h.The conclusion of ESBLs charactertion:The affinity of the blaSHV-12 type and blaTEM-1V type ESBLs against cefalexin are all the biggest in respetive hydrolysis exprements and Tazobactam:Amoxicillin 1:4 and Sulbactum:Amoxicillin 1:2 have the biggest inhibition to blaSHV-12 type and blaTEM-1V type ESBLs,respectively.