To Analyze And Probe The Result Of Oligonucleotide Microarray Of Enterohemorrhagic E.coli O157:H7 And Initialy Investigate To The Function Of O50 Island

Enterohemorrhagic E.coli is a kind of entero-pathogenic bacteria and was isolated first during breaking out of hemorrhagic enteritis in Michigan and Oregon by Riley in 1982.E.coli serotype,O157:H7,was observated by Karmali et al.,also in 1983.O157:H7 infects the human by eating the contaminated food,result in renal inadequacy,Haemolytic Uraemic Syn-drome,thrombotic thrombocytopenic purpura,and few patients died of acute and chronic renal failure.Recently,the contagious diseases caused by new pathogenic microorganism and mutated pathogenic microorganism become the serious problem which is paid close attention to by public health community.It is the internal agent that the evolution of microbiology.During the evolution of microbiology,it not only could obtain drug fast or virulence,but also could lost genes to develop its virulence.In this study,to aim at these feature about the evolution of bacteria,on the basis of the analysis of the oligonucleotide microarray,by Cluster and TreeView,classified the strains which has happened gene deletion to four types:A1,A2,B,KM. From all detected strains choosed five ones which have broken out in crowds in Xuzhou in1999 to do PCR detection.The result is:1.in these strains:Patient 21,65,143,223,248,cva120,82364, z1797,1799,1800 in O50 island were deleted;z2970,2971,2972,2973,2974,2976,2977,2978, 2979,2980 in O76 island were deleted besides Patient 248 and cva120.2.in the group of A2 strains,part of genes were deleted in O45 island,O50 island,O93 island,O108 island;there were 17 genes related metabolism,26genes related gene transfer,7 genes related structural protein,9 genes related regulation,12 genes unknown function.We found that z1797,z1799 of O50 island deleted in 15 strains which is from patients or animals in Xuzhou,Anhui,Henan and Japan in the years of 1999 and 2000;O50 island-z1824 is presumed having 31.29%identity to CNF2 in amino acid sequence in the EDL933 complete sequence notes;In the view of those two phenomenon,obviously,it is necessary to investigate the function of O50 island for us.In this study,first,to begin with z1824,I have constructed pET-30a(+)-z1824 expression vector successfully,and verified protein expressed is Z1824 by the means of sequencing,double enzyme cutting,PCR identifying,SDS-PAGE,and Western-Blot. Then,referring to the pathogenicity of necrocytotoxin,I do the cytotoxicity experiment to obverse whether Z1824 effects Hela cell.Those showed that part of Hela cells multinucleated. This effect is consistent with one of the necrocytotoxin.However,it is not so significant that we have to do more research in future.This study also provides us clue one in the fields of function of O50 island in O157:H7,at the same time,it offers us a lot of valuable information about the evolution of bacteria,and so on.