Prokaryotically Express And Purify Protein Of IntiminC280、Bfpa And Espa From Enteropathogenic Escherichia Coli O127:H6

Background:Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantilediarrhoea in developing countries. A hallmark phenotype of EPEC is theability to produce attaching and effacing (AE) lesions。All of the geneticelements required for the production of the AE lesion are encoded in a 35 kbchromosomal pathogenicity island called the locus of enterocyte effacement(LEE). The initial attachment to host cells is mediated the EspA filamentassociated with the translocation into host cells of effectors via type 3secretion（T3SS）.The intimate attachment is mediated by the outer-membraneprotein called intimin。The type IV bundle-forming pilus (BFP) is responsiblefor microcolony formation, promoting bacterium-bacterium interactions andwas proven to be a virulence factor in volunteers . The objective of our studywas to prokaryotically express and purify protein of IntiminC280、BfpA andEspA from enteropathogenic Escherichia coli O127:H6，preparing for the nextresearch on bacterin of EPEC。Objective:(1)To construct prokaryotically express system pQE30-intiminC280 ofgene IntiminC280 from enteropathogenic Escherichia coli O127:H6，as wellas gene bfpA and espA。(2)To observe the expression of recombinant prokaryotic expression plasmid pQE30-intiminC280/M15，pQE30-bfpA/M15 and pQE30-espA/M15。(3)Cheak the expression pattern of protein IntiminC280、BfpA andEspA。(4)Try to purify protein IntiminC280、BfpA and EspAMethods:(1)PCR was employed to amplify the gene intiminC280、bfpA and espAfrom enteropathogenic Escherichia coli O127:H6。Check the Correctness ofthe three amplified sequences。(2)Recombine the three amplified sequences respectively withprokaryotically express carrier pQE30，then recombinants were converted toE coli M15。(3)M15 with recombinants was induced by IPTG to expresscorresponding protein。(4)Cheak the expression pattern of protein IntiminC280、BfpA and EspAwith SDS-PAGE。(5)Purify IntiminC280、BfpA and EspA by affinity chromatography。Results:(1)prokaryotically express carrier pQE30-intiminC280/M15,pQE30-bfpA/M15 and pQE30 -espA/M15 were constructed successfully。(2)The expression of protein IntiminC280 and EspA were successfully，but the expression of protein BfpA was failed。(3)Both protein IntiminC280 and EspA expressed with two pattern：inclusionbody and soluble protein。 (4)Successfully purified protein IntiminC280 and EspA expressed withinclusionbody。Conclusion:(1)Protein IntiminC280 and EspA were outer membrane proteins,theirprokaryotically express system can be constructed easily。(2)A fragment of protein BfpA was transmembrane，its prokaryoticallyexpress system can not be constructed easily，so as its expression。(3)With His-tag，recombined inclusionbody protein IntiminC280 andEspA can be purified。...