Expression Of Enterohaemorrhagic Escherichia Coli Shiga Toxin Type Ⅰ A Subunit (STX1A) And Its Monoclonal Antibodies Preparation

Enterohaemorrhagic Escherichia coli (EHEC), is an important zoonotic pathogen, circulating around the world, including developed and deveoping countries. The infection in humans features outbreaks, highly pathogenicity, and lethality. So it poses a huge threat to human health. EHEC infection can cause severe human diseases, including bloody diarrhea, hemorrhagic colitis(HC), hemolytic uremic syndrome(HUS), and thromboric thromobocytopenic porpura(TTP). Of them, HUS can cause kidney disfunction, resulting high mortality.Shiga toxin (Stx) is one of major pathogenic factors of EHEC infection, Stx can pass through the damaged intestinal epithelial-cell into the blood circulation, binds the globotriaosylceramide (Gb3) or/and globotetraosylceramide (Gb4) recptors, and causes the damages in the intestinal, and central nervous systems. Furthermore, the high expression of Gb3 in kidney leads excessive binding of Stx, and HUS. Therefore, Stx has become the target molecule for the diagnosis and treatment of EHEC infection. In this study, we will focus on the development of monoclonal antibodies against Stx type 1 A subunit (Stx1A).Stx1A has been recombinantly cloned and expressed. A pair of primers was designed according to the published sequence in Genbank, and a PCR was run to amplify Stx1A-encoded gene. After sequencing confirmation, the gene was double digested by NdeI and Xhol enzymes, respectively, and cloned into vector pET-22b(+). The recombinant construct was transformed into E. coli of BL21 (DE3), and induced to express protein by addition of IPTG. The recombinant proteins were purified by NiSO4 conjugated affinity chromatography, and dialyzed into PBS. The identity of recombinant Stx1 A was confirmed by SDS-PAGE, Western blot and mass spectrometry analysis.Recombinant protein was sufficiently mixed and emulsified with Freund’s adjuvant, then immunized BALB/c mice three times. Spleen cells from mice immunized with Stx1 A protein were fused with a mouse myeloma cells SP2/0. Up to 6 hybridoma cell lines, called 5D3-H6,10G9-D9,4E2-H4,6E10-D11,8E7-E6, and 2F6-F8, which can secrete StxlA-specific monoclonal antibodies (MAb), have been screened out by ELISA. After cultivation and purification, the antibodies were used to characterize their binding specificity to native Stx1 by co-immunoprecipitation. Of the 6 antibodies, only 2,8E7-E6 and 2F6-F8 can specifically bind to Stxl. The antibody secretion capability of these two hybridoma cell lines did not alter after 3 months serially passage. In summary, the development of Stx1 monoclonal antibody has laid a solid foundation for the EHEC infection diagnosis.