大肠杆菌菌毛抗原K99与耐热肠毒素STⅠ融合基因的克隆与核苷酸序列测定

Cloning and Nucleotide Sequencing of Fusion Gene of K99 Antigen and Heat-stable ToxinⅠof Escherichia coli

采用PCR技术,从大肠杆菌K99中扩增出0.54kb的K99菌毛抗原基因,然后将其克隆到pUCm-T载体上,用BglⅡ鉴定K99插入的方向并测序。选择目的基因片段插入方向正确的重组质粒经BamHⅠ和SalⅠ双酶切后,通过T4连接酶与同样经BamHⅠ和SalⅠ双酶切合成的耐热肠毒素STⅠ核苷酸序列连接,通过PCR方法鉴定分析和核苷酸序列测序分析,证明插入的K99-STⅠ融合基因片段具有正确的核苷酸序列。

K99 antigen gene fragment about 0.54 kb long from Escherichia coli was amplified by PCR method and then was inserted into pUCm-T easy vector. The recombined plasmid K99-pUCm-T was studied in detail by restriction endonuclease Bgl Ⅱand nucleotide sequencing. The K99-pUCm-T and the synthetic nucleic acid frag- ment of ST Ⅰ were both cut with restriction endonuclease BamH I and Sal Ⅰ and then linked with T4 DNA ligase . The recombined plasmid pUCm-T-K99-STⅠas obtained and analysed by PCR and nucleotide sequencing. The results showed that the nucleotide sequence of fusion gene correspond to the design of experiment.