D型产气荚膜梭菌α毒素基因的克隆与高效表达

Abstract

Abstract: 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.

Key words: Clostridium perfringens; α toxin; cloning; expression

CLC Number:

S852.61⁺6.3

LI Na, WU Jian-yong, LI Jian-jun, YANG Xue-yun, WANG Deng-feng, DUAN Xin-hua, WANG Zhi-cai. Cloning and Expression of α Toxin Gene from Toxin Type D Clostridium perfringens[J]. China Animal Husbandry & Veterinary Medicine, 2012, 39(12): 10-13.

References

1 王光华,蔺国珍,郑福英,等. D型产气荚膜梭菌ε毒素基因表达及其免疫保护作用的初步研究[J].中国畜牧兽医,2012,39(3):36~41.
2 王苗利.产气荚膜梭菌α毒素蛋白在感染小鼠器官分布的检测.泰安:山东农业大学,2011.
3 王景林,王利春,吴东林,等.A型产气荚膜梭菌α毒素基因的克隆及高效表达[J].军事医学科学院院刊,2004,28(1):28~30.
4 田冬青.产气荚膜梭菌的多重PCR定型及α、β和ε毒素基因的克隆、表达和抗血清的制备.北京:中国兽医药品监察所,2007.
5 刘家森.兔产气荚膜梭菌(A型)α毒素原核表达产物免疫原性分析.哈尔滨:东北农业大学,2004.
6 许崇波. A型产气荚膜梭菌α毒素分子生物学的研究进展[J].宁夏大学学报(自然科学版),2002,23(2):188~192.
7 赵志军. A型产气荚膜梭菌α毒素基因的克隆、高效表达及其免疫原性研究.银川:宁夏大学,2005.
8 蔡玉梅.山东省四大湖(库)淡水鱼产气荚膜梭菌毒素型调查及致病机理研究.泰安:山东农业大学,2008.
9 Leslie D,Fairweather N,Pickard D,et al. Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in E.coli:Seguence and homology with a Bacillus cereus phospholipase C[J]. Mol Microbiol,1989,3(3):383~392.
10 Pugh D G. Sheep & goat medicine[M]. USA: Elsevier,2002.
11 Saint-Joanis B,Garnier T,Cole S T. Gene cloning shows the alpha toxin of Clostridium perfringens to contain both sphingomyelinase and lecithinase activities[J]. Mol Gen Genet,1989,219(3):453~460.
12 Stokka G L,Edwards A J,Spire M F,et al. Inflammatory response to clostridial vaccines in feedlot cattle[J].Journal of the American Veterinary Medical Association, 1994,204(3):415~419.
13 Titball R W,Hunter S E. Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens[J]. Infect Immun,1989,57(2):367~376.
14 Titball R W. Clostridium perfringens vaccines[J].Vaccine,2009,27:D44~D47.
15 Uzal F A,Vidal J E,McClane B A,et al.Clostridium perfringens toxins involved in mammalian veterinary diseases[J].The Open Toxinology Journal,2010,3:24~42.
16 Williamson E D,Titball R W. A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimengtal gas gangrene[J]. Vaccine,1993,11(12):1253~1256.

[1]	ZHENG Hao, LIU Mengyun, WANG Mingyu, DONG Xia, SHI Liangyu, YU Bo, YANG Yu, ZHOU Ao, CHEN Xing. Cloning, Bioinformatics and Tissue Expression Analysis of ARHGDIB Genes in Ducks [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3004-3015.
[2]	LI Zhiquan, FU Sijing, XU Shuping, GAO Mengruo, YANG Taotao, ZHANG Zhibang, LI Kai, LI Pengcheng. Cloning, Bioinformatics Analysis and Tissue Expression of HSPA5 Gene in Wuyi Black Pig [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3214-3224.
[3]	CHEN Xuke, PU Na, ZHANG Yuxia, ZHAO Wenqing, ZHAO Jiaxin, HUANG Juncheng, WEI Linying, ZHANG Yanyan, SUN Yan, BO Xinwen, WANG Zhengrong. Bioinformatics Analysis of Methionine Aminopeptidase Ⅱ of Echinococcus granulosus and Its Differential Expression in Different Stages of the Worm [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3286-3296.
[4]	ZHANG Pian, CHEN Jing, ZHANG Xiaoxiao, MAI Xiaopeng, TANG Ke, XIANG Hua, WANG Gang, LUO Shengjun, MA Huihai, YUAN Ziguo, WANG Xiaohu. Expression of GRA1 Protein of Toxoplasma gondii and Establishment of an Indirect ELISA Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3308-3320.
[5]	YANG Quan, LI Xiao, YAN Zunqiang, WANG Pengfei, HUANG Xiaoyu, GAO Xiaoli, YANG Qiaoli, GUN Shuangbao, YANG Jiaojiao. Cloning,Bioinformatics Analysis and Tissue Expression of CXCL12 Gene in Hezuo Pigs [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2482-2493.
[6]	CHU Hongen, LIU Yuan, FENG Xue, BAI Xue, YANG Mengli, LI Juan, HE Lixia, LIU Shuang, FENG Lan, MA Yun. Cloning,Bioinformatics and Tissue Expression Analysis of Bovine TGFB1 Gene [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2506-2518.
[7]	SHAN Jinyan, LIAN Kaiqi, MA Jun, LIU Yuling, NIE Zhulan, LI Binshun, PENG Renhai, WEI Jie. Cloning,Sequence Analysis and Spationtemporal Expression of Runx2b Gene in Qihe Crucian Carp (Carassius auratus) [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2717-2728.
[8]	WEI Huangsiwu, ZHANG Xingyi, HUANG Xiaohua, LIU Changjin, WU Wenjie, SHEN Zhengqiao, LUO Feng, DENG Shunzhou. Eukaryotic Expression of Porcine Rotavirus VP6 Protein and Preparation and Application of Its Monoclonal Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2750-2761.
[9]	MENG Zhaoying, JIN Huan, TU Min, HU Ge, ZHANG Zhenhua. Preparation and Identification of Rabbit Polyclonal Antibody of Chicken Complement Receptor 2 [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2772-2780.
[10]	LI Tianxiu, LI Xinpeng, DONG Xinxing, LAN Guoxiang, YAN Dawei, ZHU Jiawei. Amplification,Sequence Analysis and Tissue Expression Study of HMOX2 Gene in Lijiang Pigs [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2219-2231.
[11]	LIU Min, CHENG Huai, ZHENG Zhenzhen, ZHANG Mengsi, ZHANG Hewei, REN Jingqiang. Eukaryotic Expression and Polyclonal Antibody Preparation of N Protein of Canine Parainfluenza Virus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2287-2294.
[12]	HU Huihui, FU Panpan, LI Jie, YAN Zunqiang, GAO Xiaoli, YANG Jiaojiao, HUANG Xiaoyu. Cloning,Identification and Tissue Expression Analysis of TRIF Gene CDS Region in Hezuo Pigs [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1478-1487.
[13]	ZHANG Ying, CHEN Guangzhen, LIU Mei, LI Hongxia, YU Yan, JIA Xuebo, LI Wenqian, FAN Jianhua, MA Zhiyu. Cloning,Bioinformatics and Tissue Expression Analysis of NMB Gene in Mulard Duck [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1705-1715.
[14]	SI Wei, TAN Xiyu, XU Lingyun, LUO Tingrong, LI Xiaoning, GU Jinyan. Prokaryotic Expression of HA1 Protein of Swine Influenza Virus Subtype H3N2 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1739-1749.
[15]	MIAO Shukui, MI Xiaoyun, WEI Jie, WEI Yurong, HAILIQIEMU·Maimaitiyiming. Cloning and Bioinformatics Analysis of VP7 Gene of the Genotype G8 Bovine Rotavirus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1784-1795.

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

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments