Cloning of alpha-beta fusion gene from Clostridium perfringens and its expression.
OBJECTIVE
To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression.


METHODS
Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay.


RESULTS
The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta 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 degC, 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 alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.


CONCLUSIONS
The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.
College of Life Science, Hebei Normal University, Shijiazhuang 050016, Hebei Province, China.
Jia-Ning
JN
Bai
Yan
Y
Zhang
Bao-Hua
BH
Zhao
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology / Q000633:toxicity; D001483:Base Sequence; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000633:toxicity; D003001:Cloning, Molecular / Q000379:methods*; D003016:Clostridium perfringens / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D050939:Gene Fusion; D005798:Genes, Bacterial; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D010738:Type C Phospholipases / Q000235:genetics* / Q000276:immunology / Q000633:toxicity
D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens; D002135:Calcium-Binding Proteins; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens