产气荚膜梭菌β-毒素基因的克隆与核苷酸序列分析

Cloning and Nucleotide Sequencing of β Toxin Gene of Clostridium perfringens

为了给产气荚膜梭菌（Ｃｌｏｓｔｒｉｄｉｕｍｐｅｒｆｒｉｎｇｅｎｓ）β毒素基因工程亚单位苗和细菌毒素多价基因工程苗的研制提供基因材料，用聚合酶链式反应（ＰＣＲ）技术，从Ｂ型产气荚膜梭菌染色体基因组中扩增了９３０ｂｐ的β－毒素基因。用限制性核酸内切酶ＢａｍＨＩ和ＥｃｏＲＩ对ＰＣＲ产物进行双酶切处理，然后通过Ｔ４ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的载体质粒ｐＥＴ－２８Ｃ（＋）的多克隆位点，转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＥｃｏＲＩ双酶切分析和ＰＣＲ扩增检测，证明重组质粒ｐＥＣＢ２中含有产气荚膜梭菌的β－毒素基因。经核苷酸序列分析，明确了克隆的β－毒素基因在重组质粒中的连接向位和阅读框架是正确的。

In order to provide α gene material for prepare the gene engineering vaccine against Clostridium perfringens, β toxin gene was amplificated from genomic DNA of Clostridium perfringens type B by polymerase chain reaction(PCR) PCR product was cleaved with restriction endonucleases Bam HI and Eco RI,and inserted into vector pET 28C(+) directively,Which had been cleaved with Bam HI and Eco RI.The recombinant plasmid pECB2 was identified by restriction endonuclease analysis,PCR amplification test and nucleotide sequencing.The results showed that β toxin gene fragment of 930 bp have been cloned with a positive reading frame.