Preparation Of Quantum Dot Immunochromatographic Strip For Detecting α Toxin Of Clostridium Perfringens Type A From Rabbit

Clostridium perfringens,formerly known as Clostridium welchii,is a pathogenic bacterium that can cause infections and diseases in humans and various animals.With the prohibition of antimicrobial feed additives,acute necrotic enteritis in rabbits caused by Clostridium perfringens has become one of the important diseases that seriously threaten the development of rabbit breeding industry in China.The α toxin of Clostridium perfringens is a key virulence factor in the pathogenesis and a target protein for its post-infection diagnosis and specific treatment.This study aimed to establish a specific,sensitive and simple in vitro diagnostic method for Clostridium perfringens infection based on the isolation,identification and whole genome sequence analysis of rabbit Clostridium perfringens.The alpha toxin gene was cloned,analyzed and expressed in prokaryotic cells as recombinant CP-alpha protein(r CP-α),which was used to prepare monoclonal antibodies(Mc Ab)and rabbit polyclonal antibodies against the alpha toxin.Quantum dot-based immunochromatographic strip for detecting α toxin of Clostridium perfringens type A were trial-manufactured.Details as follows:1.A Gram-positive bacillus was isolated from the ileal contents of dead rabbits with abdominal distension by conventional microbiological method.After colony characteristics observation,16 S r RNA gene sequencing,bacterial toxin typing and ANI analysis,the isolated bacterium was identified as Clostridium perfringens type A and named TS0018.Genomics sequencing showed that the whole genome of the bacterium has 3 113 200 bp,with a GC content of 28.19%,and a total of 2 885 coding genes.The concatenation sequence was uploaded to NCBI with the accession number JALCVA000000000.In all 282 tandem repeat sequences,6 gene islands,1 prophage,and 6 CRISPRs were predicted for the whole genome of TS0018 strain.The results of protein coding gene annotation showed that TS0018 had a strong ability of metabolism and biotransformation.In addition,51 virulence genes related to mediating adhesion,invasion,nutrition/metabolism,exotoxin,extracellular enzyme and control system,and 22 resistance genes related to 8 types of drug targeted resistance and multiple drug resistance efflux pump were also detected.The phylogenetic tree of 8 housekeep genes of Clostridium perfringens showed that TS0018 strain and tumat strain,isolated from Siberian permafrost,had the closest genetic distance,and both strains were ST146.2.According to the results of virulence gene annotation,the full-length α toxin gene of TS0018 strain was cloned and analyzed by PCR,then primers were designed for PCR amplification of the mature peptide gene.After purification,digestion,transformation and PCR identification,the cloning vector and expression vector were constructed,and the αtoxin was expressed by prokaryotes.The optimal expression conditions were determined as follows: IPTG concentration of 0.8 mmol/L,temperature of 37 ℃,and time of 6 h.The r CP-α was expressed as a 60.5 k Da recombinant protein in inclusion bodies.3.Polyclonal antibody and Mc Ab were prepared by immunizing New Zealand white rabbits and BALB/c mice with r CP-α.The titer of rabbit polyclonal antibody by indirect ELISA was 1:521 000 with good specificity.In addition,the fusion rate of prepared hybridoma cells was 100%(144/144),and the positive rate was 9.28%(13/144).The antibody titer of strain 1C1 was the highest.Western blot analysis showed that the Mc Ab secreted by strain 1C1 could specifically bind to the natural α toxin.The heavy chain of the Mc Ab was Ig G2 b type and the light chain was Kappa type.The average number of chromosomes in hybridoma 1C1 strain was 99.6,and the titer of ascites prepared was as high as 1:6 553 600.4.Using carboxyl quantum dots conjugated with Mc Ab 1C1 as the labeled antibody,rabbit polyclonal antibodies as the detection line,and sheep anti-mouse Ig G as the quality control line,various materials were assembled to produce a quantum dot-based immunochromatographic test strip for detecting α toxin.The results showed that the coupling effect of Mc Ab and quantum dots was optimal at p H 7 MES buffer.The optimal concentration of sheep anti-mouse Ig G and rabbit polyclonal antibody was 0.5 mg/m L and1 mg/m L,respectively.The detection limit of the prepared quantum dot-based immunochromatography strip for r CP-α was 175 ng/m L.The cultures of Escherichia coli,Salmonella,Staphylococcus aureus and Enterococcus faecalis were all negative with good repeatability and stability.Then the detection could be completed within 15 min,providing A specific and reliable detection method for the rapid diagnosis of Clostridium perfringens type A infection in rabbits.