Cloning And Expression Of Shiga Toxin Gene And Research Of The Immunity Of Shiga Toxin

Shiga toxin is a potent toxin produced by Shigella dysenteriae I strains. The toxin has cytotoxicity, enterotoxicity, and neurotoxicity and is considered to be one of the main causes of severe side effects, such as the hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, of shigellosis. It is also an important biological warfare.The genes of Shiga toxin subunit A, subunit B, and their tandem were amplified from the genome of Shigella dysenteriae I type with the synthetic primers. The three sequences were inserted into pMD 18-T Vector and sequenced. The homogeneities of the three sequences to their corresponding sequences in GenBank are 99.5%, 100%, and 99.8%. Parameters of the proteins induced from the DNA sequences were analysed.Stx B gene achieved high expression after it was inserted into pBV220. The optimizing condition of the expression of Stx B was researched. The purity of 97.8% of Stx B was obtained through four steps, ultracentifugation, ammonium sulfate fraction, ion exchange chromatography and gel filtration. Mouse immunized with purified Stx B produced polycolonic antibody and counteracted the attack of a lethal dose of roughwrought Shiga toxin. Checken immunizied with purified StxB could produce antibody up to a high tire of 5.12 × 104 measured by ELISA.Two peptides corresponding to the protective epitopes of Shiga toxin subunit B were designed and synthesized. Peripheral blood lymphocyte was separated from human body immunized with the two peptides. Total RNA was extracted and transcriped to cDNA. The genes of V K and VH of human Ig were amplified by primers synthsised and linked by flexible linkers to form the gene repertoire of ScFv. The contraction of phage antibody repertoire and the selection of antibody of high affinity were need to be performed.