Immunoprotective Effects Of Clostridium Perfringens ?,? And ?-toxin Fusion Proteins Expressed By Bacillus Subtilis In Mice

Clostridium perfringens is an important pathogen of zoonotic diseases,which can cause necrotic enteritis,intestinal toxemia and traumatic gas gangrene in humans and animals.Because the disease caused by C.perfringens has the characteristics of rapid onset and high mortality,it has great potential threat to the animal husbandry industry.The main cause of the pathogenesis of C.perfringens is its exotoxin secretion.The species is classified into five toxinotypes?A,B,C,D and E?according to the toxins that the bacterium produces:alpha,beta,epsilon,or iota while Alpha,beta,and epsilon toxins are the main causes of disease.In the livestock industry,vaccinations are the most common prophylactic measures that are currently in use which consist of toxoids that are obtained from C.perfringens cultures.Compared with conventional toxoids,recombinant vaccines are highly targeted,stable and safe,and have a wide commercial space.They have been widely studied as a more promising immunization program.Bacillus subtilis is a highly promising foreign protein expression host,with a safe record of human and animal use as a probiotic and food additive,significant heat resistance,and relatively easy genetic and bacterial manipulation.B.subtilis,whether used to efficiently express foreign proteins or as a carrier for oral vaccines or whole-cell enzymes,exhibits unparalleled advantages of other bacteria,and is a biosafety organism with many studies and applications.Previous studies have demonstrated that Cpa^247-370 is able to confer immunity against the alpha toxin of Clostridium perfringens,while Etx^H106P is considered to be the best specific vaccine candidate,while beta toxin currently has no clear valid epitopes.The beta toxin protein secondary structure,hydrophilicity,plasticity,antigenicity,protein signal peptide and cleavage site were predicted to establish a three-dimensional spatial structure model of beta toxin protein.The online server predicted linear and discontinuous epitopes with 5consecutive residues as the minimum standard,labeled the?-toxin protein dominant epitope,and screened,intercepted and established a new protein three-dimensional spatial structure model.The recombinant plasmid pET28a-Cpb^108-305 was constructed and transformed into E.coli competent BL21 by electroporation,and the expression product was identified by Western blot analysis.The results showed that there were obvious bands at about 24 kda.Mouse immunization experiments were performed in three groups,each group was orally administered BL21-pET28a-Cpb^108-305(1 ml,10 ¹⁰ CFU/ml),BL21-Cpb solution(1 ml,10¹⁰ CFU/ml)and PBS?1 ml?.Three mice were collected from each group on the 0th,7th,14th,21st,28th and 35th day after immunization to collect blood and intestinal lavage fluid.Serum and intestinal lavage samples from immunized mice were tested by indirect ELISA to compare the immunity between the new protein Cpb^108-305 and beta toxin protein.The results indicated that the predicted screening of the new protein Cpb^108-305 was substantially identical to the beta toxin protein.In this study,a fusion protein containing protective epitopes of three toxins of?,?and?was constructed in a step-by-step manner,and a prokaryotic expression system was constructed using Bacillus subtilis.The Cpa^247-370,Cpb^108-305 and Etx gene sequences were amplified from Clostridium perfringens type B by PCR.The 74th amino acid residue?histidine?was mutated to glycine by SOE-PCR,and the 118th amino acid residue?histidine?of?toxin was mutated to proline,which was amplified and mutated by SOE-PCR.The completed three sequences of Cpa^247-370,Cpb^108-305 and Etx^H106P were recombined into a new fusion sequence Cpa^247-370-Cpb^108-305-Etx^H106P?Cpa-b-x?.The Cpa-b-x gene fragment amplified by PCR was ligated into the pHT43 expression plasmid to construct the recombinant expression plasmid pHT43-Cpa-b-x,and the accuracy of gene sequencing was confirmed.The recombinant expression plasmid was transformed into the Bacillus subtilis WB800N competent cells prepared in advance by electroporation,and positive transformants were selected from the LB plates.We induced the expression of Cpa-b-x fusion protein and identified the expressed product by SDS-PAGE and Western Blot.The results showed that there was a distinct band around about 71.15 kda,indicating that we successfully expressed the Cpa-b-x fusion protein.In order to evaluate the immune effect of the fusion protein,we further performed multiple immunoassays on the fusion protein.We randomly divided 175 6-week-old SPF mice into 5 groups and administered the same dose of oral immunization,and boosted the immunizations 10-14 d and 24-28 d after the initial immunization.Peripheral blood and intestinal lavage fluid were collected on days 0,7,14,21,28,and 35 d after inoculation,respectively.Serum antibody,intestinal mucosal antibody titer and serum cytokine levels?IL-2,IL-4 and IFN-??were detected by ELISA.The T lymphocyte transformation rate was measured by MTT colorimetry,and the peripheral blood CD4⁺and CD8⁺levels were detected by flow cytometry.After the third booster immunization,10 mice were randomly selected from each group and challenged with 10×LD50 B Clostridium perfringens,and the clinical manifestations of the mice after challenge were observed for 7 days.After one week,the protection rate of each group after the attack was calculated.The results showed that the oral immunization of Bs-pHT43-Cpa-b-x in mice can stimulate the body to produce specific antibodies,and the immunoprotective effect is significantly better than other groups.In addition,we demonstrate that B.subtilis can enhance the body's immune response to a certain extent.In this study,we predicted a protective epitope with a similar immune effect to beta toxin by predicting beta toxin and constructed a recombinant fusion protein Cpa-b-x.The study has shown that Cpa-b-x protein can effectively enhance the body's immune defense,and has a significant protective effect on mice,while Bacillus subtilis also has immunomodulatory effects on the body.This study updated the theoretical data for the recombinant study of Clostridium perfringens and provided technical support for the research of oral recombinant vaccine for Clostridium perfringens.