产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达

doi:10.16431/j.cnki.1671-7236.2015.02.019

›› 2015, Vol. 42 ›› Issue (2): 324-330.doi: 10.16431/j.cnki.1671-7236.2015.02.019

产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达

林初文^1,2, 张松林², 刘磊³, 马永彪³, 韩文瑜¹, 汪洋³, 沈志强^2,3

1. 吉林大学动物医学学院, 长春 130062;
2. 山东省滨州畜牧兽医研究院, 滨州 256600;
3. 山东绿都生物科技有限公司, 滨州 256600

收稿日期:2014-08-15 出版日期:2015-02-20 发布日期:2015-02-13
通讯作者: 沈志强 E-mail:bzshenzq@163.com
作者简介:林初文(1975-),男,山东高密人,博士生,副研究员,研究方向:预防兽医,E-mail:linchuwen@263.net
基金资助:
山东省现代农业产业技术体系羊产业创新团队项目(SATS-201226-3)

Cloning and Expression of β1 Toxin Gene from Clostridum perfringens Type B C58-1 Strain

LIN Chu-wen^1,2, ZHANG Song-lin², LIU Lei³, MA Yong-biao³, HAN Wen-yu¹, WANG Yang³, SHEN Zhi-qiang^2,3

1. College of Veterinary Medicine, Jilin University, Changchun 130062, China;
2. Shandong Binzhou Animal Science & Veterinary Medicine Academy, Binzhou 256600, China;
3. Shandong Lvdu Bio-sciences & Technology Co., Ltd., Binzhou 256600, China

Received:2014-08-15 Online:2015-02-20 Published:2015-02-13

摘要/Abstract

摘要： 利用PCR技术,从B型产气荚膜梭菌中国标准株C58-1株扩增出β1毒素基因,连接pMD18-T 载体筛选阳性克隆,然后用限制性核酸内切酶BamHⅠ和SalⅠ对其进行酶切,回收927 bp的β1毒素基因片段,将其定向克隆到载体pET-32a中,获得重组质粒pETβ927。将pETβ927转化至受体菌BL21(DE3)中,其表达产物经His-Trap FF预装柱纯化、SDS-PAGE 检测目的蛋白大小和分布及Western blotting检测其反应原性。结果表明,完整的β1毒素基因大小为1 011 bp,与GenBank发表的B型和C型产气荚膜梭菌β1毒素蛋白序列同源性达99.4%以上;SDS-PAGE结果显示重组目的蛋白在大肠杆菌中成功表达,融合蛋白大小为 54 ku,在超声波裂解上清和包涵体中均有分布,但以包涵体为主。Western blotting检测结果显示表达的重组蛋白可与特异性血清抗体发生免疫反应,表明β1毒素蛋白具有较好的反应原性。

关键词: B型产气荚膜梭菌中国标准株C58-1株; β1毒素基因; 克隆表达

Abstract: β1 toxin gene was amplified from Clostridum perfringens type B C58-1 strain by polymerase chain reaction (PCR),PCR products were connected to pMD18-T vector screening positive clones,and then cleaved with restriction endonucleases BamHⅠand SalⅠ,the 927 bp gene fragment was recovered and inserted into the same site of pET-32a vector.The recombinant plasmid pETβ927 was studied in detail by restriction endonuclease analysis and nucleotide sequencing.The recombinant plasmid could produce β1 toxin protein by SDS-PAGE.Expressed products were purified by pre-installed column of His-Trap FF,the size and distribution of the target protein were detected by SDS-PAGE,and its immunorectivity was confirmed by Western blotting.The results showed that the β1 toxin gene was 1 011 bp and the homologies with B and C type Clostridum perfringens protein sequences of GenBank were greater than 99.4%;In SDS-PAGE analysis,the fusion protein was 54 ku as expected and distributed in ultrasonic lysis supernatant as well as in inclusion bodies,but mainly existed in inclusion bodies.Western blotting analysis showed that the β1 toxin protein had a good immunorectivity with specific serum antibody.

Key words: Clostridum perfringens type B C58-1 strain; β1 toxin gene; cloning and expression

中图分类号:

Q786

林初文, 张松林, 刘磊, 马永彪, 韩文瑜, 汪洋, 沈志强. 产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达[J]. , 2015, 42(2): 324-330.

LIN Chu-wen, ZHANG Song-lin, LIU Lei, MA Yong-biao, HAN Wen-yu, WANG Yang, SHEN Zhi-qiang. Cloning and Expression of β1 Toxin Gene from Clostridum perfringens Type B C58-1 Strain[J]. , 2015, 42(2): 324-330.

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产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达

Cloning and Expression of β1 Toxin Gene from Clostridum perfringens Type B C58-1 Strain

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