摘要
霍乱毒素Ｂ亚基（ＣＴＢ）是良好的免疫佐剂和载体蛋白。本研究通过定点突变，在ＣＴＢ基因（ｃｔｘＢ）３′端终止密码前引入了限制性内切酶ＥｃｏＲＩ，构建了质粒ｐＭＣ０５。ｐＭＣ０５中ＣＴＢ与下游ｌａｃＺ′基因阅读框架相同，转化大肠杆菌后能够表达ＣＴＢ与β－半乳糖苷酶α肽的融合蛋白；所表达的融合蛋白能与ＧＭ１结合，说明融合蛋白保持ＣＴＢ的基本高级结构和生物学活性；融合蛋白能与抗－ＣＴＢ抗体结合，说明融合蛋白具有ＣＴＢ的抗原性。以上结果表明：通过将外源抗原决定簇基因融合至ｃｔｘＢ的３′端，在大肠杆菌中表达融合蛋白，构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响，构建了高效表达融合蛋白的载体－宿主系统。
Cholera toxin B subunit (CTB) is considered to be an effective adjuvant and carrier protein. In order to make gene fusions, A plasmid pMC05 was constructed by introducing an unique EcoRI restriction endonuclease site at the 3' end of CTB gene and the stop codon of CTB gene was removed at the same time so the fusion protein of CTB-β-galactosidase αpeptide could be expressed when pMC05 was transformed into E. coli , The abilities of the fusion protein to bind both ganglioside GM1 and CTB antibodies indicated that the fusion protein retains the biological function and the antigenicity of CTB. Our results show that gene fusions at the 3' end of CTB provides a new approach for the construction of engineered peptide vaccines. Our studies also show that the transcriptional terminator sequence plays an important role in the high expression of the gene fusions. Based on the results, a vector-host system was established for the high expression of fusion proteins.
关键词
霍乱毒素B亚基
基因融合
肽苗
Cholera toxin B subunit (CTB)
Gene fusions
Peptide vaccine
