Gene Fusion and Expression of Heat labile and Heat stable of Enterotoxigenic Escherichia coli Project of Chinese National Programs for High Technology Research and Development(No.863 102 07 03 05).
产肠毒素大肠杆菌的热敏肠毒素B亚基(LT-B)和耐热肠毒素(ST)基因融合及其表达

The vaccine candidate comprising the genes that code for the B subunit of the heat labile enterotoxin (LT B) and the heat stable enterotoxin (ST) of enterotoxigenic Escherichia coli (ETEC) had been constructed by recombinant genetic techniques. The 5'terminus of the gene encoding pro ST was genetically fused to the 3'terminus of the LT B gene. The pro ST gene contains mature ST sequence and pro sequence which codes for the pro region of ST precursors, and amplified by PCR from pSLM004. To reduce toxicity of the ST in vitro was substituted Leu for Ala residue at position 14 of ST by oligonucleotide directed site mutagenesis. For this construction, the expression of ST antigenicity and LT antigenicity were obtained when a five amino acid or a nine amino acid linker were included between the LT B and pro ST moieties. The LT B/pro ST fusion peptides possessed no enterotoxic activity of Escherichia coli heat stable and head labile enterotoxins,and retained the ability to bind GM1 ganglioside. More importantly,these LT B/pro ST fusion peptides were immunogenic. The preparations containing the hybrid molecule elicited special antibodies that were able to recognize native toxin in vitro.