Isolation And Characterization Of Shiga Toxin Producing Escherichia Coli And Prokaryotic Expression Of Shiga Toxin Type Ⅰ/Ⅱ Subunit Genes

Shiga toxin-producing Escherichia coli(STEC)is a zoonotic pathogen that can cause serious diseases in humans.Mainly through food,water and fresh produce contaminated with STEC.Shiga toxin(Stx)is the main virulence factor of STEC.Stx can destroy the synthesis of protein in intestinal mucosal epithelial cells,leading to cell death,mucosal shedding and eventually diarrhea.Therefore,Stx has become a targeted molecule for the diagnosis and treatment of complications of STEC infection.In this study,we intended to lay a foundation for research related to Stx targeted treatment of onset of STEC infection by cloning and prokaryotic expression of Shiga toxin type ⅠA subunit(Stx1A),Shiga toxin type ⅠB subunit(Stx1B),Shiga toxin type Ⅱ A subunit(Stx2A)and Shiga toxin type ⅡB subunit(Stx2B).In this study,a pearl fungus was isolated from calf feces and identified by 16S rRNA gene to have the highest homology with Escherichia coli STEC413.The PCR amplification with common serotype primers showed that the strain was O-antigen serotype O45.The pathogenicity test confirmed that the strain was virulent,sensitive to amoxicillin,enrofloxacin and florfenicol,and resistant to erythromycin,gentamicin and streptomycin.The Stx1A,Stx1B,Stx2A,and Stx2B four-segment genes were amplified and separated from the isolated beads by PCR,and the sequence analysis showed that the homology was more than 90%with the reference sequence.Cloning vectors pEASY-Stx1A,pMD18-T-Stx1B,pEASY-Stx2A and pMD18-T-Stx2B were successfully constructed with target gene fragments of 972bp,294bp,984bp and 294bp,respectively.Besides,prokaryotic expression plasmids of pET-28a(+)-Stx1A,pET-28a(+)-Stx1B,pET-28a(+)-Stx2A and pET-28a(+)-Stx2B were successfully constructed.After SDS-PAGE analysis,the protein sizes expressed were 38kDa,13kDa,38.9kDa and 13kDa,respectively,which were consistent with the expected sizes.Western blot test results showed that all four proteins had good immunogens.In this study,four recombinant vectors including PET-28A(+)-Stx1A,pET-28a(+)-Stx1B,pET-28a(+)-Stx2A and pET-28a(+)-Stx2B were successfully constructed.Western blot analysis showed that the expressed proteins of STX1A,Stx1B,Stx2A and Stx2B could react with specific antibodies with good reactivity,which laid a foundation for further research.