绿脓杆菌外毒素A的Ⅰ+Ⅱ区基因的克隆与表达

Cloning and expression of the exotoxin A domain Ⅰ + Ⅱ structural gene fragmant of Pseudomonas aeruginosa

绿脓杆菌外表素Ａ是由６１３个氨基酸残基组成的单链杀细胞毒素；利用ＰＣＲ技术直接从绿脓杆菌染色体ＤＮＡ中扩增了外毒素Ａ的Ⅰ＋Ⅱ区的结构基因，通过酶切鉴定、ＤＮＡ序列分析，证实了克隆的基因片段为外毒素Ａ的结构基因；重组表达质粒经诱导表达后ＳＤＳ－ｐＡＧＥ及Ｗｅｓｔｅｒｎ ｂｌｏｔ证实了表达的产物为外毒素Ａ分子。对外毒素Ａ的Ⅰ＋Ⅱ区结构基因的克隆表达为疫苗研制、毒素类似物的制备以及导向药物的构建打下了基础。

Pseudomonas exotoxin A (ETA ) is a 6 13-residue cytocidal protein. In this report , a 1 . 25kb fragment of exotoxin A Domain Ⅰ+Ⅱ structural gene of Pseudomonas aeruginosa was amplified form chromosomal DNA of Pseudomonas aeruginosa by using PCR technology. The amplified fragment was cloned into expression vector pET-3a directly. The recombinant plasmids were screened and were determined with restriction enzymes digestion. The cloned fragment was also sequenced with dideoxynucleotide termination method and automated DNA sequencing by using PCR primer to guide the sequencing. The result of the sequencing identified that the cloned gene was a fragment of ETA. SDS-PAGE assay and western blot confirmed that the expression products are ETA-related polypeptides.