Expression Of ?-?-? Toxin Fusion Protein From Clostridium Perfringens And Analysis Of Its Immunogenicity

Clostridium perfringens,also known as C.WELCHII,a rod-shaped,gram-positive pathogen,is widely found in nature.It is a highly dangerous zoonosis pathogen,causing food poisoning in humans and a variety of animals,traumatic gangrene or gas gangrene,secretory,necrotizing and hemorrhagic enteritis,enterotoxemia,dysentery etc..It is characterized by acute onset,short course of disease and high mortality,which is a great threat to human health and animal husbandry.The Clostridium perfringens is non-invasive and its pathogenicity is mainly due to the action of exotoxin.So far,18 Clostridium perfringens have been identified.According to the production of four major toxins?CPA,CPB,Etx,Iota?,Clostridium perfringens are divided into five types:A,B,C,D and E.It makes difficult for conventional drugs to cure diseases caused by Clostridium perfringens,because these diseases which are characterized by acute onset and short course are often not found in time.The most widely used vaccines are polyvalent vaccines which are composed mainly of inactivated bacteria or toxoid or a mixture of them.However,these polyvalent vaccines have some disadvantages such as complex antigenic components and few effective antigenic components.Recombinant vaccines may be a solution.Recombinant vaccines may be a solution.Previous studies have shown that CPA^247-370,which is the C terminal of Clostridium perfringens toxin,can confer high protective rate in mice;rETX^H106P,a toxin with H106P mutation,has significantly reduced toxicity and has considerable to be immunogenicity,and has the potential to become a candidate protein for Genetic Engineering Vaccine.In the meanwhile,previous studies have shown that CPB¹08-30508-305 which has a concentrated epitope,can reduced toxicity and have no significant difference in induced protective level from inactivated toxin-like vaccine.The result is due to the following reasons:?1?,toxin is widely found in five toxin types;?2?Clostridium perfringens is often caused by a combination of toxins;?3?studies have shown that the protective effect of recombinant multivalent toxins is due to univalent toxins.Therefore,we use genetic engineering methods to express these three toxins(CPA^247-370-CPB^108-305-rETX^H106P)in tandem to prevent the infection of various toxin types.In this study,we successfully constructed Cp-abx(CPA^247-370-CPB^108-305-rETX^H106P).P.pastoris eukaryotic expression system has post-translational modification functions such as glycosylation,fatty acylation,protein phosphorylation.What effects of these functions in expressing of recombinant toxin Cp-abx(CPA^247-370-CPB^108-305-rETX^H106P)and its immunogenicity.Meanwhile,E.coli prokaryotic expression system has been widely used as a tool for the expression of foreign genes.In this study,both Pastoris Eukaryotic system and E.Coli was used to express Cp-abx(CPA^247-370-CPB^108-305-rETX^H106P).The safety of the recombinant protein was evaluated in vitro and in vivo,and the recombinant protein was found to be safe and stable.The recombinant protein was fully mixed with Freund's incomplete adjuvant as immunogen to evaluate the immune effect of mice and screen the suitable expression system.In addition,Pine Pollen polysaccharide has immunomodulatory effect.Pine Pollen polysaccharide was taken orally during mice immunization to evaluate its effect.The Cp-abx fusion gene was optimized by the company according to the codon preference of E.Coli and Pichia Pastoris,respectively.The genes were amplified by PCR,cloned into Blunt3 Vector,and then transformed into E.coli DH5 clone strain and amplified.After PCR and sequencing were identified correctly,PET-28a?+?empty plasmid and Blunt3-cp-abx double-enzyme were digested with NcoI and Xho I endonuclease respectively,and were linked by T4 DNA ligase and transformed into DH5 vector for identification.After identification,the plasmid was amplified,extracted and transformed into BL21?DE3?natural competence by thermal stimulus method,and identified as pET-28a-Cp-abx.The empty plasmid was also transferred to the BL21?DE3?Natural competence and named Pet-28a-vector as a negative control.After the pPIC9 secretory expression plasmid was selected in Pichia Pastoris,and the fusion fragment was correctly linked to the pPIC9expression plasmid,it was linearized by single enzyme digestion with the empty plasmid by restriction enzyme SacI,and was transferred into Pichia Pastoris GS115 Natural competence by electrotransformation.The positive transformants were identified correctly and named pPIC9-cp-abx.The empty plasmid was named pPIC9-vector as the negative control.The fusion protein was induced by IPTG before the logarithmic growth phase.After 0,1,2,3,4,5and 6 hours of induction,the supernatant was centrifuged at 4°C,the supernatant and the cell were collected and broken up by ultrasound.Similarly,pPIC9-Cp-abx was induced to expressing by methanol,and the supernatants and cells were collected by centrifugation at4°C in 12000rpm.SDS-PAGE analysis showed that there were 71.15 KDa molecules in the Gel chromatography of the two expression systems.The objective band was confirmed by Western Blotting analysis,and the target band in SDS-PAGE corresponded to the target protein The recombinant Pichia Pastoris was induced to express the protein mainly in the supernatant and expressed in secretion.The expressed protein was purified on nickel column,and then SDS-PAGE showed only 71.15 KDa single target band.In addition,the expression of the fusion protein in Pichia Pastoris was lower than that in E.coli.The purified protein was mixed with IFA adjuvant and the concentration of protein was measured by BCA method.180 SPF mice aged 5-6 weeks were divided into 6 groups,which were inoculated with PPIC9-Cp-abx,pPIC9-Cp-abx and orally administered PPPS,pET28a-cp-abx,pET28a-cp-abx and orally administered PPPS,commercial vaccine?sheep triple quadruple vaccine?and phosphate buffer solution control group?PBS?.Every week,We measured antibody levels,peripheral blood lymphocyte,and peripheral blood CD₄+and CD₈+T lymphocyte.In addition,we measured the levels of cytokines?IL-2,IL-4,IFN-??in the peripheral blood and small intestine,and evaluated the protective effect of the recombinant protein by the anti-virus protection test.The results showed that the protective effect of recombinant protein expressed by Pichia Pastoris and E.coli was 100%?6/6?as good as that of commercial vaccine,and was significantly better than that of other groups,the recombinant fusion protein group that did not take pine pollen polysaccharide orally also showed a higher protection rate of 83.3%?5/6?.In this study,the protective epitopes with similar immune effect to the toxin screened by the laboratory were linked with the effective antigenic epitopes of the combination,after codon optimization,the protein was expressed in E.coli and Pichia pastoris respectively.The results showed that the two proteins were safe and non-toxic,and could induce the immune defense of mice.In addition,Pine Pollen polysaccharide has a certain immune enhancement,to a certain extent,enhanced the body's immune response.In addition,the expression level in E.coli was higher than that in Pichia Pastoris,but there was no significant difference in the induced protection level.We therefore recommend E.coli as the preferred expression host for the fusion protein expression,with the potential to be used as a Clostridium perfringens vaccine.This study provides a theoretical basis for the study of new Clostridium perfringens vaccines.