Study On Expression Of Tir-C Of Enterohemorrhagic E.Coli O157:H7in Escherichia Coli And Lactobacillus Acidophilus

l.Background and purposeEnterohemorrhagic Escherichia coli (EHEC) O157:H7is a pathogenic microorganism mainly through the alimentary tract,which can cause hemorrhagic colitis(HC),hemolytic uremic syndrome (HUS) and thrombotic thromobocytopenie porpura (TTP) and even lead to death,the mortality of the infection rate was0%～10%.Since Riley first reported the hemorrhagic enteritis caused by EHEC O157:H7,the infection of EHEC O157:H7has become a global public health problem and has been a serious threat to public health.Therefore,the prevention and control of EHEC O157:H7infection is very important.At present,there is still no effective methods to control the EHEC O157:H7infection.EHEC O157:H7is sensitive to most antibiotics,but when Escherichia coli O157:H7was killed by antibiotics,it can release Shiga toxin,which increases the risk of patients complicated with HUS.So it is extremely urgent to develop effective vaccine to prevent and treat the infection of EHEC O157:H7.The pathogenicity of EHEC O157:H7is mainly related to the adhesion and colonization and the toxin of the bacterial.Translocation intimin receptor(Tir) O157:H7is an important virulence proteins.It is secreted by the EHEC Type III secretion system(TTSS) and inserted into the epithelial cell plasma membrane.As the receptor of intimin which is an integral outer membrane protein of EHEC,Tir combinated with intimin can cause actin cytoskeletal rearranged and form attaching and effacing(A/E) lesion,it plays an important role in the process of colonization and infection of EHEC O157:H7.Therefore,blocking intimin and Tir combination is significant to avoid A/E lesion in the infection first stage.In this study,Tir C-terminal(marked as Tir-C)was amplified by PCR considering the result of Bioinformatics analysis of Tir,then Tir-C was cloned into into the vector pMD19-T and PET-30a(+),lastly,the recombinant plasmid(designed as PET-30a(+)-Tir-C)was identified by PCR,sequencing and digested by restriction endonucleases.The positive recombinants were transformed into E.coli BL21/DE3and induced by IPTG to express the Tir-C.The Tir-C protein was detected by SDS-PAGE and identify the antigenic of the recombinant protein preliminarily by Western blot. The target gene and expression vector PMG36e were digested by Hind Ⅲ and XbalI,then linked with T4DNA ligase, the recombinant plasmid PMG36e-Tir-C was transformed into Lactobacillus acidophilus by electroporation.The recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHEC O157:H7was identificated by Gram staining、PCR、double enzyme digestion SDS-PAGE and Western blot. The aim of the experiment is to explore the most effective vaccine for preventing EHEC O157:H7after immunization with Tir-C protein、the recombinant Lactobacillus acidophilus or the recombinant Lactobacillus acidophilus combined with Tir-C protein.2.Methods2.1Bioinformatics analysis of TirThe DNA and protein sequences of Tir were achieved from NCBI website. Protein analysis was done with InterProScan and the B cell epitope of Tir was analysised by two different B cell epitope analysis softwares.2.2Prokaryotic Expression of Tir-C Gene 2.2.1Constructionof the recombinant cloning vector of Tir-CTir C-terminal (Tir-C) was amplified by PCR considering the result of Bioinformatics analysis of Tir,then Tir-C was cloned into into the vector pMD19-T, the recombinant cloning vector pMD19-T-Tir-C is identified by PCR, sequencing and digested by restriction endonucleases.2.2.2Construction of the recombinant expression vector of Tir-CThe recombinant cloning vector pMD19-T-Tir-C and expression vector plasmid PET-30a(+) were digested by Nde I and Xho7,then linked and the recombinant plasmid was identified by PCR,sequencing and double enzyme digestion.2.2.3Identification、expression and Purification of Tir-CThe PET-30a(+)-Tir-C recombinant was transformed into E.coli BL21/DE3and induced by IPTG to express the Tir-C.The Tir-C protein was detected by SDS-PAGE and purified by Ni-IDA agarose.The antigenic of the recombinant protein is preliminarily identified by Western blot using the6×His monoclonal antibody as first antibody,and horse radish peroxidase sheep-mices IgG as the second antibody.2.3Construction of the recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHEC O157:H72.3.1Amplification of Tir-C geneAccording to the base of Tir-C gene and the restriction enzyme sites of plasmid PMG36e, specific primers was designed.Tir-C gene was successfully amplified with the primers.2.3.2Construction of recombinant plasmid PMG36e-Tir-CThe target gene and expression vector PMG36e were digested by Hind Ⅲ and XbaI,then linked with T4DNA ligase.2.3.3Construction of recombinant Lactobacillus acidophilus strainsThe recombinant plasmid PMG36e-Tir-C was transformed into Lactobacillus acidophilus by electroporation.The recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHEC O157:H7was identificated by Gram staining、PCR、double enzyme digestion、SDS-PAGE and Western blot. 3.Results3.1Bioinformatics analysis of TirThe length of the Tir is1677bp,encoding558amino acids totally,with the initiation codon ATG and the termination codon TAA;The protein contains three structural and functional domains,namely Tir N terminal,C terminal and Intimin binding region M;The protein includes twenty linear B-cell epitopes with Bepipred analysised and twenty-six linear B-cell epitopes with ABCPred analysised.According to the result of Bioinformatics analysis,1000bp-1674bp Of Tir(marked as Tir-C) is taked as the target fragment.3.2Prokaryotic Expression of Tir-C GeneTir-C gene was amplified from EHEC O157:H7strain Guangzhou882364strain, the length of the Tir-C is675bp.The recombinant plasmid pMD19-T-Tir-C is successfully constructed according to the identification of plasmid PCR,Nde I and Xho I double enzyme digestion and sequencing;The recombinant cloning vector pMD19-T-Tir-C and expression vector plasmid PET-30a(+) were digested by Nde I and Xho I,then linked with T4DNA ligase.The recombinant plasmid was successfully constructed with identification of PCR, sequencing and double enzyme digestion; The Tir-C protein was successfully expressed when the PET-30a(+)-Tir-C recombinant was transformed into E.coli BL21/DE3and induced by IPTG.Tir-C was purified by Ni+affinity chromatograghy.Concentration of the purified protein is about500ug/ml and a unique band was detected with the relative molecular mass of approximately24KDa by Western blot.3.3Construction of the recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHEC O157:H7Tir-C was successfully amplified by PCR, The recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHEC O157:H7was successfully constructed according to the identification of Gram staining、PCR、double enzyme digestion、SDS-PAGE and Western blot.4.Conclusion 1、The1000bp-1674bp of Tir(marked as Tir-C)was successfully achieved as the target fragment considering the result of Bioinformatics analysis of Tir,the length of Tir-C is675bp.2、The recombinant cloning vector of Tir-C pMD19-T-Tir-C was successfully constructed;The recombinant expression vector of Tir-C PET-30a(+)-Tir-C was successfully constructed. A unique band was detected with the relative molecular mass of approximately24KDa by Western blot,which shows the Tir-C protein had immunoreactivity to some extent.3、The recombinant Lactobacillus acidophilus expressing the Tir-C gene of EHECO157:H7was confirmed by Gram staining、PCR、double enzyme digestion、 SDS-PAGE and Western blot.