Cloning, Modification And Expression Of Heat-labile Enterotoxin Gene From E.coli

Escherichia coli heat-labile enterotoxin (heat-labile enterotoxin, LT) was the intestinal toxicity secreted by Enterotoxigenic Eshchirichia coli (ETEC). LT was composed of subunit A (LTA) and subunit B, the former has toxicity; the latter formed five rings which can bind GM1 ganglioside on the membrane of eukaryotic cell. LT can cause diarrhea in human and other mammals. In addition to toxicity, LT was also able to trigger humoral immunity and cellular immunity, so it has the strong immunogenicity and adjuvanticity in mucous membrane and has important medical value in the study of mucosal immunity and in vaccine development.In order to prepare LT proteins at large scale with immunocompetence, the genes encoding of heat-labile enterotoxin A subunit (LTA) and the B subunit (LTB) were amplified with PCR method from the genomic DNA of Enterotoxigenic Escherichia coli strain 44815. The amplified LTA and LTB genes were inserted into pMD18-T vector to construct pMD18-T-LTA and pMD18-T-LTB recombinant plasmids, respectively. The positive clones were identified through the blue-white screening system and double-enzyme digestion(Nco I and Xho I), vertified by sequencing. The LTA gene was modified by changing Ser63 to Lys63 with Site-directed mutagenesis on the pMD18-T-LTA recombinant plasmid. LTAK63 and LTB genes were transfered into the pET-20b (+) prokaryotic expression vectors respectively through Nco I and Xho I sites and construct the prokaryotic expression vectors of pET-20b-LTAK63 and pET-20b-LTB. Recombinant expression vectors of LTA and LTB were transformed into host bacteria Bl21 respectively for preparation of engineering bacteria. The expression products were detected by SDS-PAGE and the interest protein was separated and purified by nickel-agarose beads. In order to construct LTAK63 gene eukaryotic expression vector, the LTAK63 gene with NheI and ApaI sites was amplified by PCR using pMD18-T-LTAK63 plasmid as a template. The gene was ligased with pcDNACD5sp by NheI and ApaI sites to construct the recombinant plasmid, pcDNACD5sp-LTAK63. To identify whether LTAK63 gene with human CD5 signal peptide can express in eukaryotic cells, HEK 293T cells were applied to transiently express LTAK63 gene with the calcium phosphate transfection method.The experimental results showed that the LTA gene amplified by PCR was consistent with the Gene Bank published sequence and the homology was 99%. Four bases of LTA gene were changed, but only one base results amino acid substitution from glutamic acid to Lysine. Therefore LTA gene has polymorphism. LTB gene amplified was in coincidence with the published in GenBank. Sequence results showed that Ser63 of LTA was changed to Lys63 successfully. The constructed prokaryotic expression vectors, pET-20b-LTAK63 and pET-20b-LTB, were correct by identification with Xho I and NcoⅠdigestion. The two proteins (MW of 28 kDa (LTA) and 14kDa (LTB)) were detected in shock–release sulution by SDS-PAGE, which indicated that the two interest proteins were expressed as secretive form. Purified protein by nickel adsorption showed the single band by SDS-PAGE. All illustrated that the LTAK63 and LTB were mediated by signal peptide Pel , can make the LTAK63 and LTB expressed and secreted to the periplasm, rather than existing in form of inclusion body, so the expressed protein has activity. and separation and purification were easy. Western blot results showed that there was a clear hybrid band at about 34.8KDa, but at the corresponding location of empty vector there was no hybrid band. Therefore the eukaryotic expression vector pcDNACD5sp-LTAK63 was constructed successfully, the secretive expression for LTA63 gene was achieved in eukaryotic cells.