Prokaryotic Expression,purification And The Immunological Characteristics Of Some Proteins From EHEC O157:H7

Purpose:Enterohemorrhagic Escherichia coli(EHEC)is one of the main causes of global foodborne disease,EHEC O157:H7 has been reported as a pathogen since 1982 and it has caused several outbreaks in China and worldwide,sporadic cases are often been reported in various clinical journals and reports.EHEC infection has brought significant economic losses to human health and the food industry.Because there is no way to prevent or special treatment of EHEC O157:H7,understanding the infection and pathogenic way of EHEC O157:H7 will be important to avoid the outbreak of EHEC and improve the treatment.it will also be important theoretical guidance for the development of innovative medicines,the design of treatment plan,the development of effective treatment,vaccine formulation of prevention and control measures.Escherichia coli infection mainly depends on a large number of virulence factors,and many of them have been investigated such as Intimin,Tir and the effector proteins of type III secretion system.EHEC O157:H7 transport its virulence proteins into host cells through type III secretion system,which interaction with host cell,mediates host actin aggregation,and causes an attaching-and-effacing(A/E)lesion.However,it is not clear how the signal transduction pathway in this process works.It is of great significance to elucidate the pathogenesis of EHEC O157:H7 by understanding the proteins related to attaching-and-effacing(A/E)lesion.The purpose of this study is to predict the EHEC O157:H7 type III secretion system of secreted proteins of EHEC O157:H7 by screening and identification,the Z2322,Z2340 and other 5 protein gene was cloned,expressed and purified.analysis the humoral and cellular immune responses of Z2322,Z2340 and other 3 proteins in mice through animal experiments to further clarify the immune response type(Th1/Th2)of the stimulus induced in mice.Lay the foundation for further studies of how the Z2322,Z2340,Z2691,Z3322 and Z3955 proteins work in the pathogenesis of EHEC O157: H7 and development of new vaccines.Method:Based on the EHEC O157: H7 EDL933 strains,LipoP and other six softwares are used to predict whether Z2322,Z2340 and other five proteins exist signal peptide,predict if it is outer membrane protein or inner membrane protein and their subcellular localization.Analyze their existing situation and the conservatism of the nucleotide and amino acid.Based on the genome of Escherichia coli O157:H7 EDL933 strain,the gene fragments of Z2322,Z2340 and other 5 proteins were amplified by PCR,and the restriction sites BamHI and Xho I were introduced.Double digested PCR products were ligated into pET-22b(+)and other 3 expression vectors,The resulting constructs were transformed into E.coli BL21(DE3),the expression of the fusion proteins are induced by IPTG,and the expression style of fusion proteins are identified.the successfully expressed protein with His tag were purified by Ni affinity column.Preparing mouse anti-serum,and the immunogenicity was detected by ELISA.Blood samples were collected from the tail vein in immune mice at 21,28,35,42 days.To evaluate the efficacy in actively immunized mice,the titers of IgG antibody and its subtypes(IgG1,IgG2 a,IgG3)were determined by ELISA.the immune response type(Th1/Th2)of stimulus induced in mice was analyzed.The immunized mice were infected with EHEC O157:H7 EDL933 strain,and the protective effects were detected.After the attack,the feces of mice were collected to measure the amount of live bacteria,determine the effects of proteins on bacterial colonization.Result:1.LipoP,Lipo,SignalP 4.1 predict that there is no signal peptide in Z2322,Z2340 and other 5 proteins.Via the analysis of TmHmm and Bomp,these proteins do not have ?-helix and ?-barrel structure.Through analysis of subcellular localization software PSORTb 3.0.2 and CELLO v.2.5 found that these proteins may be secreted to extracellular.2.Cloned Z2322,Z2340 and other 5 genes and constructed the recombinant plasmid.E.coli BL21-pET-28a-Z2691,E.coli BL21-pET-30a-Z2322 and other 8 proteins are expressed after induced by IPTG.In which E.coli BL21-pET-28a-Z2691 and other 4 proteins are expressed solubly in the supernatant;E.coli BL21-pET-30a-Z2322 and other 4 proteins are expressed in the form of inclusion bodies in precipitation.3.different methods are used to purified proteins expressed in different forms,and the molecular weight of protein was predicted by SDS-PAGE.ELISA method was used to detect the titer of Z2322,Z2340,Z2691,Z3322 and Z3955 antiserum.The titer of Z2340,Z2691 and Z3955 antiserum detecting through ELISA were 62500 EU,Z2322 was 12500 EU,Z3322 was 2500 EU.Z2322,Z2340,Z2691,Z3322 can stimulate mice to produce high titer of IgG1 and IgG2 a,and the IgG antibody subtypes are mainly composed of IgG1,Z3955 can stimulate the body to produce an immune response dominated by Th2 type immunity.4.the immune protection test showed that Z2340,Z2691 and Z3955,Z2322 can pro duce a good protective effect on mice and reduce mortality.Z3322 can prolong t he time of death,produce a protective effect on mice.This is consistent with the r esults of the ELISA method for the detection of serum titers in mice stimulated with each protein.the amount of bacteria experimental results show that,bacteria s are not detected after 6d,7d,9d,9d after O157:H7 attacking in Z2322,Z2340,Z2691,Z3955 protein immued mice.The group of Z3322 protein immued mice detect ed less bacteria than that of control group.indicating that Z2322,Z2340,Z2691,Z3955 have a certain effect on reducing the survival rate of EHEC O157:H7 while Z3322 is not obvious.