Construction Of Recombinant Lactobacillus Casei Expression Alpha Toxin Protein Of Clostridium Perfringens And Immunogenicity Analysis

Clostridium perfringens is one of the pathogenic bacterial resulting in Necrotic enteritis(NE), food poisoning, as well as enterotoxaemia.The pathogenic factor is exotoxin secreted by bacterial itself, based on different types of exotoxins, it can be divided into five different serotypes, which are A,B,C,D and E serotype.Among them C perfringens alpha toxin(CPA) is the shared virulence factor of all types of Clostridium perfringens, also the main toxin produced by type-A bacterial. This toxin combines the enzymatic activity of PLC and sphingomyelinase, both can dissolve the main part of cell membrane—phosphatidylcholine and sphingomyelin, and destroy the integrality of cell membrane, then cause cell lysis, thus it's one type of toxin with high cytotoxicity, haemolyticus, mortality, skin necrosis and so on. Diseases are caused by the production of bacterial in intestinal system and absorption of bacterial in alimentary tract system in the period of mass reproduction.By considering the absorption of toxin and mass reproduction ability in intestinal tract system, designation of stimulation to intestinal tract immune system, which can block the invasion into first immune system, is very important for protecting this disease. This research uses Lactobacillus casei ATCC 393(L.casei 393)as bacterial carrier to transport antigens, and uses gene fragment encoding C. perfringens alpha toxin as targeted gene. The research use gene fragment mutated in the site of C. perfringens alpha toxin(mutate genes encoding 68 Histidine residues into Glycine residues in the level of nucleotide, causing loss of cytotoxicity. However remaining the completeness of antigenicity) and insert into Lactobacillus plasmid, thus constructing the recombinant Lactobacillus system with surface expression and secretion expression of CPA toxin by Clostridium perfringens. By the expression and transmit of exogenous antigens and the characteristics of Lactobacillus in the area of intestinal-adhesive, bile-endurable, pancreatin-endurable, as well as colonization and probiosis to the intestinal tract, it can functionally stimulate immunosystem of intestinal tract system, thus preventing poisoning of Clostridium perfringens by making it into oral-vaccine medicines.Using pET-30b-αplasmid, which encodes Clostridium perfringensα-toxin, as template, also in terms of plasmid fusion site and the tendency toward the usage of Lactobacillus casei codon, we developed a pair of primer by oligo6.0 software, then enlarge into 924bp target gene, after that, we cloned that gene into pMD18-T simple vector, then under enzyme digestion, PCR identification, the recombinant plasmid was named pMD18-T-α. This recombined plasmid was digested by both Sph Ⅰand XhoI, then getting target gene, connecting with plasmid pPG-1 and pPG-2, which was digested using the identical endonucleases.After that,competent status of L.casei 393 is electro-transformed, then we selected positive cloning, treating it with enzyme-digestion, PCR and sequencing, the recombined plasmid is name as pPG-1-αand pPG-2-α, the recombined Lactobacillus casei is named as pPG-1-α/L.casei393 and pPG-2-α/L.casei393.Inducing the expression of target proteins of these two recombined Lactobacillus casei, we use 1% lactose as inducer.The results of SDS-PAGE showed that around 34KD recombined protein(cell-surface type and cell-secretion type) is expressed, and it's corresponding to our expectation. Western blot showed that, the expressed protein can be detected by type A rabbit Clostridium perfringens alpha toxin, indirect immunofluorescence and western blot showed that the expressed proteins can be detected on the surface of Lactobacillus casei. After inducement, we take the supernatant of the bacterial and do the SDS-PAGE, around 34KD proteins are expressed, corresponding to the expected values. Western blot showed it has the same antigenic specificity with natural toxin proteins.To explore the application value of recombinant of Lactobacillus casei with Clostridium perfringens alpha toxin, this experiment uses BALB/c mouse as test for immunology response. The procedure is: orally inject 109 number of bacterial, then immune every 2w, four times immunities in total, with each immunity period lasting for 3 days, and immunize BALB/c mouse one time per day. In the mean time, we vaccinate Lactobacillus casei with blank plasmid and PBS into another BALB/c mouse as negative comparison.After immunization, we measure the secreted anti-alpha toxin IgA taken from mice excrement, vagina fluid, tear sample and the level of specific anti-alpha toxin IgG in mice blood serum. The result of ELISA showed that: In mice excrement, vagina fluid and tear sample, we detect specific sIgA, meanwhile, in mice serum, we also detect high level of anti-serum, that means the recombinant of Lactobacillus casei can induce local immune response, as well as stimulate Humoral immune response in mice body. Besides, we toxify the mice, which is in its fourth period of immunization, with alpha toxin, so as to determine the immune function of recombinant to antibody against alpha toxin in mice body, the result also showed that the antibody against alpha toxin, which is expressed after transformation of recombined Lactobacillus casei, can suppress the natural alpha toxin activity produced by type-A Clostridium perfringens. After collecting the mice serum and performing the neutralization experiment, we confirmed the serum gained the anti-alpha toxin activity. The analysis tells us the secretedly recombined Lactobacillus casei reaches better immunity level, thus providing referential foundation for the field in which we orally vaccinate live bacterial as plasmid and get antibody.For the first time in this field, we construct recombinant of Clostridium perfringen alpha-toxin into Lactobacillus casei surface-expression and secretion system, we successfully get the expression of recombined protein in food microorganism.The antibody, which is expressed in the immunized animal, has a high level of immunity, it makes us to believe that using recombined Lactobacillus casei as oral vaccine has a potential application value, and it lays a firm foundation for exploring next new types of alpha-toxin oral vaccine.