Cloning of α-β fusion gene from Clostridium perfringens and its expression

• Published in: World journal of gastroenterology : WJG Vol. 12; no. 8; pp. 1229 - 1234
• Main Authors: Bai, Jia-Ning, Zhang, Yan, Zhao, Bao-Hua
• Format: Journal Article
• Language: English
• Published: United States College of Life Science, Hebei Normal University, Shijiazhuang 050016 , Hebei Province, China 28.02.2006; Baishideng Publishing Group Co., Limited
• Subjects: Animals; Bacterial Toxins - genetics; Bacterial Toxins - immunology; Bacterial Toxins - toxicity; Base Sequence; Basic Research; Blotting, Western; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - immunology; Calcium-Binding Proteins - toxicity; Cloning, Molecular - methods; Clostridium perfringens - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli - genetics; Gene Expression; Gene Fusion; Genes, Bacterial; Genetic Vectors; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - physiology; Type C Phospholipases - genetics; Type C Phospholipases - immunology; Type C Phospholipases - toxicity; 基因克隆; 基因表达; 细菌感染; α-β fusion gene; Cloning and expression; Clostridium perfringens
• ISSN: 1007-9327; 2219-2840
• DOI: 10.3748/wjg.v12.i8.1229

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β fusion gene was constructed and transformed into BL21（DE3）. The recombinant strain BL21（DE3）（pZCPAB） was obtained. After the recombinant strain BL21（DE3）（pZCPAB） was induced by 42℃, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.