Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β₂ toxin (cpb₂) and atypical-cpb₂ (aty-cpb₂), analyze the epidemiological characteristics and genetic polymorphism of the cpb₂ of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb₂ of 188 Clostridium perfringens strains were examined by PCR; the cpb₂ sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb₂-library based on 110 strains carrying the cpb₂ were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb₂ (con-cpb₂) and aty-cpb₂. Results: The specificity of PCR assay for the cpb₂ and aty-cpb₂ was verified. The PCR results for cpb₂ amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb₂, 94 types A strains carried aty-cpb₂, 6 types A strains carried con-cpb₂, and 7 types F strains carried aty-cpb₂. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb₂ toxin was developed, and the previous PCR method for detecting aty-cpb₂ was improved. aty-cpb₂ is the primary gene encoding of β₂ toxin. There is a significant nucleotide sequence variance between the various cpb₂ genotypes.
Humans ; Clostridium perfringens/genetics* ; Clostridium Infections ; Bacterial Toxins/genetics* ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Genetic

We constructed a recombinant strain BL21 (DE3) (pETST3LTBalphabeta) including ST1-LT(B)-alpha-beta fusion gene via molecular technology. The SDS-PAGE and Western blotting indicated that the ST1-LT(B)-alpha-beta fusion protein was highly expressed in Escherichia coli and the molecular weight of the fusion protein was about 110 kD. The recombinant strain was induced in different concentrations of lactose and different aeration rate. The optimal culture conditions in 20 L fermentor were 1% inoculation (V/V), initial aeration 5 L/min, 0.03 mol/L lactose addition 3 hours after inoculation, and increased the aeration to 12.5 L/min for the following 6 hours. The fusion protein was about 38.53% of total cellular protein. It was nontoxic, immunogenic and protective against enterotoxigenic E. coli and Clostridium perfringens infection. The constructed recombinant strain BL21 (DE3) (pETST3LTBalphabeta) could serve as a candidate vaccine strain against diarrhea of piglet.
Animals ; Bacterial Toxins ; biosynthesis ; genetics ; Calcium-Binding Proteins ; biosynthesis ; genetics ; Clostridium perfringens ; genetics ; Dysentery ; prevention & control ; veterinary ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; immunology ; Escherichia coli Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transformation, Bacterial ; genetics ; Type C Phospholipases ; biosynthesis ; genetics ; Vaccines, Synthetic ; immunology