Construction And Expression Of Fusion Protein Of Alpha Beta2Epsilon Beta1Toxin Of Clostridium Perfringens In Recombinant Lactobacillus Casei And Its Immunogenicity Analysis

Clostridium perfringens is the major pathogenic bacterium which can cause traumatic gas gangrene, alimentary toxicosis, enteritis necroticans and enterotoxaemia.Its causative agent is thirteen secretory exotoxin of clostridium perfringens, α、β、ε and ι are main toxins. Type A, TypeB, TypeC, TypeD and Type E are five serotypes of clostridium perfringens, according to differennt types of exotoxin. Alpha-toxin that is the main toxin of clostridium perfringens of A type and is the common virulence factor of all clostridium perfringens. It can destroy integrality of cellular membrane and cell disruption, so, it is to result in haemolyticus, cytotoxicity, fatal, cutaneous necrosis and so on. Betal toxin is a necrotizing and lethal toxin, which is main pathogenic factors of red dysentery of piglets with high morbidity and mortality. Beta2toxin generated by the B type and C type, as one of main virulence factors of Clostridium perfringens type C, is toxic and lethal, it can produce necrotizing enteritis and hemolytic necrosis and can cause a variety of livestock and poultry enteritis and enterotoxemia. Epsilon toxin generated by type B and D type bacteria, is the main toxin type D of Clostridium perfringens. Preparing immunogen of a variety of toxins to resist to heterologous toxin attack have practical significance to effective prevention of Clostridium perfringens diseases.In this research we utilize Escherichia coli BL21(DE3) as antigen presenting vector of α-β2-ε-β1fusion protein to research the immunogenicity.Alpha beta2toxin fragment was clonied from the recombinant plasmid pET-30b-α-β2which was constructed by our aboratory, and alpha toxin mutant (histidine residues encoding the68th nucleotides mutated as glycine residues), β2toxin mutation (cysteine residues encoding234th nucleotides mutated as glycine residues) lost their toxicity but remain immunoge. Epsilon and betal genes were obtaind by PCR, and were sequentially connected with the alpha beta2fragments to construct α-β2-ε-β1fusion gene and then was cloned into pProHTa expression vector, and transformed into Escherichia coli BL21(DE3).The recombinant plasmid was named as pProHTa-α-β2-ε-β1and the recombinant Escherichia coli was named as pProHTa-α-β2-ε-β1/BL21. In further study, recombinant Lactobacillus casei pPG-2-α-β2-ε-β1/L. casei393was constructed with the Lactobacillus casei ATCC393as antigen presenting vector.The recombinant Lactobacillus casei was induced by1%lactose, and SDS-PAGE results shows that about126ku fusion protein was expressed, which protein size corresponds with the theoretical value. Western blot analysis showed that the expression of protein can be tested with antiserum of alpha toxin of Clostridium perfringens type A, which shows good specificit.As live vaccine, the recombinant Lactobacillus casei are explored in terms of the application value.BALB/c mouse were used as test animal to do immune experiment. Immunization programs:the mouse were orally inoculated with109CFU/mL live bacteria of pPG-2/L.casei393and pPG-2-α-β2-ε-β1/L.casei393and PBS as negative control. Total three immunizations were performed every2weeks. The immunization was performed once a day and each immunization time were successive three days. After immunization, mice vaginal mucus, feces and tear samples were tested to determine secreted IgA and serum specific IgG of anti α-β2-ε-β1toxin. ELISA test results show that, there were most specific sIgA in mice feces, more vaginal mucus then tear samples and the samples were also detected high level of specific IgG in mice serum at the same time, toxin on The immune mice were challenged10days after third immunization and the results show that, antibody generated in mice can resist attack of natural toxin of Clostridium perfringens of type A and type B. Mice immunized with the recombinant Lactobacillus casei pPG-2-α-β2-ε-β1/L.casei393were induced local immune response and humoral immuneresponse. The recombinant Lactobacillus casei can stimulate the body to produce systemic and specific antibody which have good immune protection effect on mice. To sum up, as oral vaccine, the recombinant expression system of Lactobacillus casei has some potential value in application, which lays the foundation for developing new oral vaccine of Clostridium perfringens.