| Lactic acid bacteria(LAB)are a group of gram-positive bacteria that can generate lactic acid by ingesting large amounts of carbohydrates in the environment.In recent years,lactic acid bacteria have received more and more attention due to their properties of probiotics,and have been recognized as probiotic microorganisms by the Food and Agriculture Organization of the United Nations(FAO)and the World Health Organization(WHO).Lactic acid bacteria have a large genome capacity,high biological safety,and strong resistance to gastric acid and bile.After oral immunization,they can adhere to the surface of the intestine and continuously stimulate the body to produce systemic humoral immune responses,mucosal immune responses,and cellular immune responses.It is a living carrier Ideal for expressing foreign proteins in vaccines.Porcine viral diarrhea is an important epidemic disease that has harmed the swine industry and caused great economic losses to the world’s swine industry.Most of the existing commercial porcine viral diarrhea vaccines are inactivated vaccines or attenuated vaccines,which have the disadvantages of slow antibody production,low antigen content,and the risk of reverting to strong virulence.Therefore,the development of a safe and efficient new type of porcine viral diarrhea multi-linked oral lactic acid bacteria live carrier vaccine is of great significance for the prevention and control of porcine viral diarrhea.This study aims to develop a novel recombinant live vector vaccine of lactic acid bacteria(PEDV,TGEV,and PDCo V),and to conduct a preliminary evaluation of their immune effects,which provide a theoretical foundation and technical platform for the development of lactic acid bacteria oral vaccines against porcine viral diarrhea infection.1 Construction of triple recombinant lactic acid bacteria of porcine viral diarrheaPorcine viral diarrhea is an important epidemic disease that has harmed the pig industry and caused great economic losses to the world’s swine industry.At present,inactivated vaccines and attenuated vaccines are mainly used to prevent the disease.Due to the prone mutation of the Spike(S)genes of PEDV,TGEV,and PDCo V and other factors,the immune protection efficacy of the current commercial vaccines is reduced.Therefore,the development of new and efficient vaccines has become an urgent problem to be solved.Based on the enterotropism of the three viruses,part of the S genes of PEDV,TGEV and PDCo V were selected as target genes in this study.The NICE expression system was used to express part of the S proteins of PEDV,TGEV,and PDCo V respectively.In addition,the three segments of proteins are connected by two flexible junction peptides to achieve the fusion expression of the three viral protective antigens.The results of the SDS-PAGE,Western-blot,and indirect immunofluorescence test showed that the single and fusion proteins expressed by the recombinant lactic acid bacteria were localized on the surface of bacterial and showed good reactivity.In addition,the expression conditions of the recombinant lactic acid bacteria were optimized,and the optimal induction conditions were as follows:the concentration of the inducer nisin was 20 ng/m L,the temperature was 37℃,and the time was 5h.Through the analysis of the basic biological characteristics and genetic stability of the recombinant lactic acid bacteria,it was found that the target gene could stably exist in the lactic acid bacteria without affecting the growth of the host bacteria.2 Immune efficacy evaluation of triple recombinant lactic acid bacteria in porcine viral diarrheaThe use of lactic acid bacteria to express target proteins and deliver antigens to the mucosal immune system,so that the expressed target proteins are continuously produced in the mucosa and then stimulate the body to produce a mucosal immune response is the current research hotspot in the field of oral vaccines.To evaluate the immunological efficacy of trivalent recombinant lactic acid bacteria for porcine viral diarrhea on animals,ICR mice were used as an animal model,and the recombinant lactic acid bacteria expressing the fusion proteins P(PEDV partial S protein),T(TGEV partial S protein),D(PDCo V partial S protein)and PTD(PEDV,TGEV and PDCo V partial S protein)were immunized by intragastric administration,and the immune efficacy of the recombinant lactic acid bacteria was studied.The results show that the recombinant lactic acid bacteria could effectively stimulate the body to produce humoral immunity,mucosal immunity,and cellular immunity.The levels of serum-specific Ig G and s Ig A antibodies were significantly increased in mice after intragastric immunization.The levels of IFN-γand IL-4 cytokines were significantly higher than those in the control group.The proliferation of CD3~+,CD4~+,and CD8~+T lymphocytes was significantly increased,and CD3~+,CD4~+,and CD8~+T lymphocytes were stimulated.The results showed that the recombinant lactic acid bacteria expressing the protective antigen of porcine viral diarrhea could stimulate the immune response of the body,and could be a potential candidate for an oral vaccine for porcine viral diarrhea.In conclusion,the recombinant lactic acid bacteria expressing the protective antigens of PEDV,TGEV,and PDCo V were successfully constructed in this study.The recombinant lactic acid bacteria can effectively induce good humoral,mucosal,and cellular immune responses in mice,providing technical support for the research of a new oral vaccine against porcine viral diarrhea. |
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