铜绿假单胞菌外毒素A基因DNA片段的克隆及序列测定

Cloning and sequences analysis for a partial gene of exotoxin A of Pseudomonas aeruginosa

目的 建立含有铜绿假单胞菌外毒素A基因DNA片段的克隆株。方法 根据已报道的序列设计铜绿假单胞菌外毒素A基因的特异引物 ,用PCR方法从标本扩增出预期大小的DNA片段 ,并将该片段纯化后与T载体连接 ,转化到大肠埃希菌 ,筛选出含有插入片段的克隆。结果 阳性克隆株经DNA序列测定 ,证实与已报道的铜绿假单胞菌外毒素A基因DNA序列 ,在399个碱基中仅有 3个碱基不同 ,但编码的氨基酸完全一致。结论 成功构建含有铜绿假单胞菌外毒素A基因的克隆 。

Objectives To obtain the cloning of partial DNA in exotoxin A gene of Pseudomonas aeruginosa Methods According to the reported sequence of exotoxin A of Pseudomonas aeruginosa ,a pair of specific primers was designed and synthesized.The expected DNA segment with 399 base pair was amplified by PCR from a sample.The PCR product was purified,cligated into T vector and cloned in Escherichia coli to screen the clones containing the inserted segment.Results By sequence determination the sequence of positive cloning was confirmed to be similar to the reported DNA sequence of exotoxin A gene of Pseudomonas aeruginosa except 3 mismatched base but the sequence of coded amino acid was completely consistent.Conclusion All the results proved that we were successing in cloning the partial gene of exotoxin A of Pseudomonas aeruginosa .It will be available for preparation of batch of probes and stable and reliable positive reference for detection of Pseudomonas aeruginosa by PCR method.