牛产气荚膜梭菌α-ε毒素基因的融合表达及其纯化  

Fusion expression and purification of bovine Clostridium perfringens α-ε fusion toxin gene

作　　者：尹辉[1] 赵达[1] 陈为宏[1] 高佳滨[1] 乔波[1] 陈楠楠[1] 胡旭[1] 梁鸿儒[1] 姜东君[1] 王鹤[1] 朱战波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2015年第3期261-265,共5页Chinese Journal of Biologicals

摘　　要：目的融合表达产气荚膜梭菌α毒素基因和ε毒素基因的融合基因α-ε,并进行纯化。方法利用PCR技术,从A型产气荚膜梭菌C57-8株基因组DNA中扩增出α毒素基因的部分基因片段,插入到质粒p ET28a-ε上,构建含α-ε融合毒素基因的表达质粒p ET28a-α-ε,转化至E.coli BL21(DE3)plys S感受态细胞中,IPTG诱导表达,表达的重组蛋白经Ni-Agarose His标签蛋白纯化试剂盒纯化后,进行Western blot鉴定。结果重组质粒p ET28a-α-ε经PCR及双酶切鉴定证明构建正确,与预期的基因序列重合率为99%。表达的重组蛋白相对分子质量约78 000,主要以包涵体形式存在,表达量占菌体总蛋白的22.7%,纯度为91.2%,可与小鼠产气荚膜梭菌多抗血清特异性结合,具有良好的反应原性。结论成功构建了重组质粒p ET28a-α-ε,并在E.coli BL21(DE3)plys S中表达了重组α-ε融合蛋白,该蛋白具有良好的反应原性,可作为预防牛肠毒血症基因工程亚单位疫苗的候选抗原。Objective To express and purify the fusion gene α-ε of bovine Clostridium perfringens. Methods Partial αtoxin gene fragment was amplified from the genomic DNA of C57-8 strain of Clostridium perfringens type A by PCR and inserted into plasmid p ET28a-ε. The constructed recombinant plasmid p ET28a-α-ε was transformed to competent E. coli BL21（DE3）plys S and induced with IPTG. The expressed recombinant protein was purified by Ni-Agarose His tag purification kit and identified by Western blot. Results Both PCR and restriction analysis proved that recombinant plasmid p ET28a-α-ε was constructed correctly, of which 99% of the gene sequence was consistent with that expected. The expressed recombinant protein, with a relative molecular mass of about 78 000, mainly in a form of inclusion body, contained22. 7% of total somatic protein, and reached a purity of 91. 2%, which showed specific binding to polyclonal antisera against mouse C. perfringens, indicating a good reactogenicity. Conclusion Recombinant plasmid p ET28a-α-ε was successfully constructed, and recombinant α-ε fusion protein was expressed in E. coli BL21（DE3） plys S. The expressed fusion protein showed good reactogenicity, which might be used as a candidate antigen for recombinant subunit vaccine against cattle enterotoxemia.

关 键 词：产气荚膜梭菌 Α毒素基因 ε毒素基因 融合表达 纯化 

分 类 号：S855.12[农业科学—临床兽医学] Q786[农业科学—兽医学]