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

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).

采用基因重组技术构建了表达产肠毒素大肠杆菌（ Ｅ Ｔ Ｅ Ｃ） 的耐热肠毒素（ Ｓ Ｔ） 基因和热敏肠毒素 Ｂ 亚基（ Ｌ Ｔ Ｂ） 基因融合抗原的疫苗候选株。将 Ｓ Ｔ 基因的５’端与 Ｌ Ｔ Ｂ 基因的３’端连接，并置于同一阅读框。编码 Ｓ Ｔ 的基因是通过 Ｐ Ｃ Ｒ 从ｐ Ｓ Ｌ Ｍ００４ 质粒中扩增得到的，含有 Ｓ Ｔ 的ｐｒｏ 序列（ 其编码 Ｓ Ｔ 前体的ｐｒｏ 区域） ，并应用寡核苷酸定点突变技术将编码 Ｓ Ｔ 的第１４ 位氨基酸残基发生突变，使 Ｓ Ｔ 的第１４ 位氨基酸残基 Ａｌａ 突变为 Ｌｅｕ 。在所构建的结构中，于 Ｌ Ｔ Ｂ 和ｐｒｏ Ｓ Ｔ 之间分别插入了不同长度的氨基酸 Ｌｉｎｋｅｒｓ 。表达的融合多肽同时具有 Ｓ Ｔ 和 Ｌ Ｔ Ｂ 的抗原性，并保留结合 Ｇ Ｍ１ 神经节苷脂的能力，且无 Ｌ Ｔ 和 Ｓ Ｔ 的生物毒性。表达的融合蛋白免疫动物，能诱导产生相应的特异性抗体。

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.