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]. , 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]	WEI Mingbang, BIANBA Qiongda, XIAO Qingqing, DUAN Mengqi, CHAMBA Yangzom, SHANG Peng. Cloning,Bioinformatics and Expression Analysis of CDKN1B Gene in Tibetan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2196-2206.
[2]	XIAO Peng, SHANG Jianghua, YANG Chunyan, LI Mengqi, DUAN Anqin, MA Xiaoya, FENG Chao, HUANG Chenqian, ZHANG Bo, ZHOU Jinchen, WEI Kelong, ZHENG Wei, ZHENG Haiying. Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2380-2387.
[3]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[4]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[5]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[6]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[7]	WANG Haorui, YU Baojun, YANG Jiayi, WEI Rong, FU Xi, WANG Chuanchuan, ZHANG Di, LI Desheng, CAI Zhengyun, GU Yaling, ZHANG Juan. Bioinformatics Analysis and Tissue Expression of DERA Gene in Jingyuan Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1764-1773.
[8]	PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.
[9]	ZHUO Na, ZHU Zhongwu, LI Jing, HU Shixiang, WANG Jianchang, LIN Hua, CHEN Chaolin, HAN Diangang, AI Jun, CHEN Peifu. Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1959-1970.
[10]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[11]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[12]	TAO Qinghua, HUANG Caiyun, CHEN Chao, LIU Wei, SUN Ningning, LI Ang. Cloning and Tissue Expression Difference Analysis of LIF Gene in White Muscovy Duck [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1296-1306.
[13]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[14]	LIU Hongrun, ZHU Siran, FENG Lingli, ZHANG Kun, YAN Gang, WANG Yubin, ZHANG Shuai, JIANG Shan, XU Di, LAN Ganqiu, LIANG Jing. Cloning,Bioinformatics Analysis and Tissue Expression Localization of CDO1 Gene in Large White Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1352-1363.
[15]	ZHANG Zhewei, DAI Xiaotong, WEI Zudan, CHEN Yunpeng, GAN Jing, BEI Weicheng. Study on Prokaryotic Expression and Immunogenicity of OmpW and TbpA Proteins in Swine Pasteurella multocida [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1489-1498.

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