D型产气荚膜梭菌α毒素基因的克隆与高效表达

Cloning and Expression of α Toxin Gene from Toxin Type D Clostridium perfringens

为建立产气荚膜梭菌(Clostridium perfringens)α毒素(cpa)克隆表达方法,应用PCR方法从羊源D型产气荚膜梭菌中扩增cpa成熟肽基因序列,将其插入pET-28b载体中,构建重组表达载体pET-28b-cpa;通过PCR扩增、双酶切和测序方法对重组载体进行鉴定及序列分析,然后转入BL21(DE3)pLysS中诱导表达;用SDS-PAGE检测目的蛋白大小及分布,Western blotting方法检测其反应原性。结果表明,所扩增的cpa基因大小为1110 bp,与GenBank参考序列同源性为99%以上;SDS-PAGE分析结果表明,目的蛋白大小为41.2 ku,与预期大小一致,在超声波裂解上清和包涵体中均有分布,但主要以包涵体为主,两者分布均具有和天然毒素相似的反应原性。

To establish the method for cloning and expression of Clostridium perfringens α toxin gene（cpa）,the cpa gene was amplified by PCR from toxin type D strain,then was inserted into pET-28b to construct pET-28b-cpa.Identified by PCR,restriction enzyme digestion and sequencing methods,the recombinant plasmid was transformed into BL21（DE3） pLysS and induced to express by IPTG. The size and distribution of the target protein were detected by SDS-PAGE,and its reactionogenicity was confirmed by Western blotting. The results showed that the cpa gene was 1110 bp and the homology with reference sequence of GenBank was greater than 99%.In SDS-PAGE analysis,the target protein was 41.2 ku as expected and distributed in ultrasonic lysis supernatant as well as in inclusion bodies,but mainly existed in inclusion bodies.Both of them showed similar reactionogenicity to native α toxin.