Clone And Sequencing Of The Gene Sequences Related To Multidrug Resistance In Shigalla Spp.

Shigella spp.is pathogen of bacillary dysentery. The annual number of Shigella infections all over the world was estimated to be 165 million, a large part of which occurred in developing countries.More than half of the infection affects children under 5 years of age. shigellosis is the third gravest contagious disease of all contagious diseases in Chair and the emphasis of the prevention and cure of contagious diseases.Recently, the phenomenon of drug resistance in shigella spp.is becoming increaseingly serious:not only drug resistance is wide spread, but also the strains with multidrug resistance increase gradually, which is a big challenge to prevention and cure of bacillary dysentery.There are two main orgins of drug resistance:horizontal gene transfer and induced by antibacterials. Horizontal gene transfer means a method,mediated by movable genetic factors such as plasmids,transposons,integrons and gene islands, for bacteria to obtain or spread resistance between intraspecies or interspecies. Resistance induced by antibacterials means bacteria may get resistance though variance of certain genetic mechanism under antibacterial environment.In order to understand the relationship between the emerge of bacterial resistance and the change of genome under the induction of antibiotics, our study group once obtained a inducted multidrug-resistant strain by the antibiotic induction, and was successful in constructing the library of suppression subtractive hybridization, and preliminarily screened some gene fragments related to induced multidrug-resistant strain.On basis of the study before, the study analyzed different gene sequences between the sensitive strain and the induced multidrug-resistant strain, and analyzed how the resistances to happen and the probable positions, which will provide scientific basis and skill methods for illuminating the molecule mechanism of induced multidrug resistance in shigella spp.and the detection of drug resistance. 1 Resuscitation and identification of strains:to resuscitate the strains kept in-80℃refrigerator by streaking, and in order to make sure that the strains are reliable, biochemistry and serology methods were carried out to identigy these strains.2 Extraction of DNAs:genomic DNAs were extracted from YD and Z23 and plasmids DNAs from positive control strains,pMD18-T-X(Y16A11,Y16B3,Y19C3,Y19C1,Y19C4,Y16C4,Y16B8).3 Polymerase chain reaction(1)Plasmids DNAs of the positive control as template and nested PCR primers were used for amplification of related gene sequences.(2) Primers were designed by the software(Primer Premier 5.0), according to gene sequences finished (Y16A11,Y16B3,Y19C3,Y19C1,Y19C4,Y16C4,Y16B8),and related gene sequences are amplified from YD and Z23's genomic DNAs.(3)The products were detected by 1.5% agarose gel electrophoresis.4 Clone of related gene:after the products of PCR were purified, they were conjugated with pMD19-T vector, transformed into DH5 a competent cell,and then positive recons were screened by ampicillin resistance and blue and white blot screening method. And positive clones were identified.5 Sequencing and blast:sequencing was finished by a biological company. Nucleotide sequences were searched and analyzed.6 Dot blot hybridization:related gene sequences(Y16A11-Y,Y16A11-Z,Y16B3,Y19C3,Y19C3-Z,Y19C1,Y19C4,Y16C4,Y16B8) were labeled,and then were hybridized with DNAs of positive clones,YD and Z23.1 The products of nested PCR:positive control strains pMD18-T-X(Y16A11,Y16B3,Y19C3,Y19C1,Y19C4,Y16C4,Y16B8) were used as template. sequences (449bp,481bp,317bp,1075bp,298bp,413bp,298bp) were amplified.2 The products of PCR:Y16A11-Y(310),Y16B3(320bp),Y19C3-Y(124bp),Y19C1(422bp),Y19C4(193bp),Y16C4(227bp),Y16B8(225bp) were amplified from YD and Y16A11-Z (554bp) and Y19C3-Z (388bp) were amplified from Z23.3 The clones of gene sequences amplified:it is successful to obtain positive clone strains of Y16A11-Y,Y16A11-Z,Y16B3,Y19C3-Y,Y19C3-Z,Y19C1,Y19C4,Y16C4 and Y16B8.4'Identification of sequences amplified:the products of germ liquid PCR and zymase cutting were in accordance with expectation.5 Sequencing and blast: Y16A11-Y(310bp):a part of plasmid pBS512₅ Y16A11-Z(554bp):fatty acid metabolism transcriptional regulator FadR sequence homology of Y16A11-Y and Y16A11-Z is low. Y16B3(320bp):being divide into two sections(113 bp and 218 bp),the former is conserved hypothetical protein and the later is transposase. Y19C3-Y(124bp):no homologous sequences was found. Y19C3-Z(388bp):putative multidrug-efflux transport protein. Sequence homology of Y19C3-Y and Y19C3-Z is very low. Y19C1(422bp):multidrug efflux system component Y19C4(193bp):the sequence from122 to 193 is part of plasmid pBS512₅. Y16C4(227bp):HsdS, type I site-specific deoxyribonuclease. Y16B8(225bp):a part of it is plasmid pBS512₅.6 T he products of dot blot hybridization:all the signals that explorers (Y16B8, Y16A11-Y,Y19C3-Y,Y16C4,Y19C4) hybridized genomic and plasmid DNA of YD strengthen, and the difference of hybridization signals between genome and plasmid is small;the signal that explorer (Y16B3) hybridized only YD genomic DNA enhance; the signals that explorers (Y16A11-Z,Y19C3-Z) hybridized genomic and plasmid DNA of YD and genomic DNA of Z23 is strong, but the signals that them hybridized plasmid DNA of Z23 is weak; the signals that explorer(Y19C1) hybridized any DNA is weak.1 It is confirmed that the genomic differences are existed between the induced multidrug resistant strain and sensitive strain of shigella spp.. 2 Multidrug resistant strain acquired may result from gene mutation and genetic recombination in shigella spp..3 Multidrug rsistance gene sequenaces acquired may occur in chromosomes,and may occur in plasmids or other movable genetic elements,such as transposition, integron and box gene system,bacterial invader.