Construction of Escherichia coli LTKA63 mutant and possible mechanism of immunoadjuvant activity of its recombinant

To construct detoxified mutant LTKA63 of the heat-labile enterotoxin subunit A(LTA) gene of Escherichia coli and its prokaryotic expression system,and to study the mechanism of mucosal immunoadjuvant activity of the recombinant LTKA63(rLTKA63).The LTA gene from E.coli strain 44815 genome DNA was amplified by high fidelity PCR.Site mutation technique was applied to construct the mutant LTKA63.Two cloned genes were sequenced after T-A cloning.Then the prokaryotic expression system of LTKA63 gene was constructed by using pET-15b without His-tag and its expressed product was identified with SDS-PAGE.The pathways activating T lymphocytes of rLTKA63 were determined with Three Color Flow Cytometry.And amplification fragment of LTA gene with the expected size was obtained from E.coli strain 44815 genome DNA template.In comparison with the reported LTA gene sequences, the nucleotide and amino acid sequences of the cloned LTA gene showed the similarities of 99.03%～99.61% and 98.33%～99.22%,respectively.The mutant LTKA63 with correct sequence was obtained through the site mutation of the cloned LTA gene and expression output of rLTKA63 was approximate 10% of the total bacterial proteins.After co-incubation with LTKA63,the Th1 and Th2 lymphocytes were increased with 21.9% and 36.2%,respectively,compared to those of the negative controls.In conclusion,the prokaryotic expression system of LTKA63 gene was successfully established in this study and the expressed rLTKA63 can activate both Th1 and Th2 lymphocytes.