Construction Of Recombinant Lactococcus Lactis Vector With S1 Gene Of Variant Porcine Epidemic Diarrhea Virus And Immunization In Mice

Porcine epidemic diarrhea?PED?is a kind of acute intestinal infectious disease caused by Porcine epidemic diarrhea virus?PEDV?,which is characterized by vomiting,diarrhea,loss of appetite and severe dehydration.PED infection in piglet is100%.The mortality rate of piglets within one week can be as high as 80%¹00%.In2010,a large outbreak of PEDV infection occurred again in China.After identification and analysis,this epidemic strain is mainly composed of multiple base deletions and insertions in the S1 region of PEDV S gene,accompanied by a large number of point mutations,which causes the existing vaccine unable to provide immune protection,has brought great losses to the pig industry.Therefore,the study of new and efficient vaccines is necessary.S protein is the main structural protein of PEDV,which is divided into S1 region and S2 region.S1 region is an immunodominant region,which participates in the identification and binding of specific receptors,contains multiple neutralizing epitopes,and is often used as a candidate protein in the study of PEDV genetic engineering vaccine.PEDV is mainly colonized in the intestine.Therefore,only stimulating the body to produce mucosal immunity can prevent the occurrence of the disease.In order to achieve a good mucosal immune response,S1 gene was synthesized from CH/JLCC/2011 mutant strain?Sequence ID:JQ638920.1?published by the NCBI.Flag sequences and NcoI and SacI restriction sites were added to both ends of the sequence to clone the PEDV S1 gene.The vector pET-28a-S1 and the expression vector pNZ8149 were double-digested with NcoI and SacI.The purified product of the gene and the expression vector were ligated and electrotransformed into the host strain NZ3900 and screened with Elliker's medium to obtain positive recombinant LactococcuslactispNZ8149-S1/NZ3900.Therecombinantbacteriawere anaerobically allowed to stand at 30?in M17 medium and induced with nisin at a final concentration of 1ng/mL.After SDS-PAGE and Western-blot analysis,the target protein was expressed at a size of about 89 kDa.The results of IFA showed that the green fluorescent of pNZ8149-S1/NZ3900 was observed on the surface of the positive recombinant bacterium,and no green fluorescence was observed on the empty vector pNZ8149/NZ3900,indicating that the recombinant protein expressed by Lactococcus lactis was colonized on the surface of the cells and had reactiongenicity.In order to explore the effect of recombinant Lactococcus lactis expressing PEDV S1 protein on the level of immune response in vivo,BALB/c mice were selected as experimental animals in this study.The mice were randomly divided into four groups:pNZ8149-S1/NZ3900 group,pNZ8149/NZ3900 empty vector control,NZ3900 host bacteria and PBS control group.Each mouse was orally immunized with1.3×10¹⁰ CFU of Lactococcus lactis or PBS of 200?L and immunized in three separate immunization intervals of two weeks with three immunizations each for three consecutive immunizationsSerum,feces,cecum and small intestine contents,spleen,mesenteric lymph node and PP node were collected at different time.sIgA,IgG and the levels of IL-4 and IFN-?were detected by Elisa,splenic lymphocyte proliferation by MTT,and the activation of T lymphocytes by flow cytometry.The results showed that the levels of sIgA and IgG antibodies in pNZ8149-S1/NZ3900 immunized mice were significantly higher than that in pNZ8149/NZ3900 empty vector,NZ3900 host bacteria and PBS control group?p<0.01?,but no significant difference was found between pNZ8149/NZ3900,NZ3900 and PBS control groups.The results of splenic lymphocyte proliferation showed that 5ug/mL S1 antigen of PEDV significantly promoted the proliferation of splenic lymphocytes?p<0.01?.The results of IL-4 and IFN-?test showed that the recombinant bacteria could stimulate the mice to produce Th1/Th2 mixed-type cellular immune response.The results of flow cytometry showed that pNZ8149-S1/NZ3900 recombinant bacteria could promote the cellular immune response at a certain level.In summary,this study successfully constructed the expression of recombinant Lactococcus lactis pNZ8149-S1/NZ3900.The animal experiments confirmed that the recombinant can improve the mucosal,humoral and cellular immunity in mice,enhance immunity,and provide a new way for the study of new oral vaccine of PEDV.