Construction And Analysis Of Induced Immune Response Of Expressing VP4 Of Porcine Rotavirus With The Auxotrophic Lactobacillus

Porcine rotavirus（PoRV）is an important pathogen causing porcine viral diarrhea.Rotavirus infection has obvious enterotropism,so intestinal mucosal immunity is the first line of defense against pathogen invasion.Lactic acid bacteria（LAB）,an important probiotic with the characteristics of acid resistance,bile salt resistance,immune adjuvant function,the intestinal adhesion and colonization function,could stimulate the intestinal mucosal to generate an immune response.Based on this,this study uses intestinal probiotic LAB as a live bacterial carrier to express and deliver the protective antigen VP4 of PoRV.This study tried to achieve the purpose of reasonable prevention and treatment of PoRV infection by combining the nonspecific antibacterial and anti diarrhea effects of LAB with the specific immune advantages against PoRV.In this study,the alanine racemase gene（Alr）in porcine Lactobacillus paracasei HLJ-27（L.paracasei HLJ-27）was knocked out with the CRIPSR-Cas9^D10Agene editing system,generating an auxotrophic strain△HLJ-27.On this basis,the neutralizing protective antigen VP4 of PoRV was inserted into the thymidine synthase（thy A）site of the genome and a auxotrophic strain L.paracasei VP4/△HLJ-27 were constructed with stably expressing foreign protein,realizing the artificial control for the auxotrophic strain and laying the foundation for the establishment of food-grade selectable markers.After that,mice and piglets were orally immunized with recombinant strain VP4/△HLJ-27,and the humoral immune response,mucosal immune response and cellular immune response were evaluated.Because PoRV and other pathogens are mostly mixed infection or secondary infection,leading to the death of piglets,this study chose to monitor the changes of antibody level of oral immune recombinant bacteria within 56 days instead of challenge protection test to evaluate its immune effect.The main research results as follows:1.Construction of auxotrophic strain△Alr HLJ-27The Alr gene of porcine L.paracasei HLJ-27 genome was knocked out by gene editing plasmid pLCNICK-DA1,and the alanine auxotrophic strain△Alr HLJ-27 was constructed.Firstly,the gene editing plasmid pLCNICK-DA1 was electro-transformed into L.paracasei HLJ-27competent cells and the recombinant strain was identified by genomic PCR.The results showed that there were two bands of about 2700 bp（before knockout）and 1500 bp（after knockout）,indicating that the CRIPSR-Cas9^D10Asystem successfully edited the genome,and the efficiency was 87.5%.After further purification,the mutant strain△Alr HLJ-27 was obtained.Then,the D-alanine demand of mutant strain L.paracasei△Alr HLJ-27 was detected and the results showed that the recombinant strain could resume growth only when D-alanine was added externally or transferred into the complementary plasmid carrying Alr gene,indicating that the Alr gene auxotrophic strain was successfully constructed.In addition,the growth characteristics,genetic stability,plasmid elimination,cell morphology,high temperature tolerance,acid resistance and bile salt resistance of the auxotrophic strain△Alr HLJ-27 were further detected.The results showed that there was no significant change compared with the parent strain.2.The auxotrophic strain VP4/△HLJ-27 expressing the VP4 gene of PoRV was constructedPoRV VP4 gene was inserted into the thy A site of L.paracasei HLJ-27 genome using CRIPSR-Cas9D10A system,and a auxotrophic strain VP4/△HLJ-27 expressing VP4 gene of PoRV was constructed.Firstly,three sgRNAs sequences were designed based on the thy A site,and the gene editing plasmid was constructed.After electrotransfer into the competent cells of the auxotrophic strain△Alr HLJ-27,the results of genomic PCR showed that there were two specific bands,1849 bp before knockout and 2657 bp after insertion,indicating that the CRIPSR-Cas9^D10Asystem successfully edited the genome,and the genome editing efficiencies of the three sgRNAs were 66.7%,45.8%and 91.6%,respectively.After further purification,the mutant strain VP4/△HLJ-27 was obtained.Then,the D-alanine demand of mutant strain VP4/△Alr HLJ-27 was detected and the results showed that the recombinant strain could resume growth with exogenous addition of D-alanine,which was in line with the characteristics of auxotrophic.The results of Western blot and ELISA showed that the VP4 gene of recombinant strain VP4/△Alr HLJ-27 was expressed.In addition,compared with the parent strain,the growth characteristics,genetic stability,plasmid elimination,cell morphology,high temperature tolerance,acid resistance and bile salt resistance of the recombinant strain did not change significantly.3.Immune response induced by oral immunization of animals with recombinant strain VP4/△HLJ-27Mice were orally immunized with the recombinant strain VP4/△HLJ-27,and the feces,intestinal mucus,vaginal lavage fluid and serum of immunized mice were collected.With the PoRV as coating antigen,the results of indirect ELISA showed that the levels of SIg A and Ig G in mice were significantly increased,and the titer of serum neutralizing antibody was 1:32.Cytokines IL-4,IL-2,IL-10,IL-12,IL-17 and IFN-γin serum were detected and the results showed that the recombinant bacteria could significantly stimulate mice to produce Th1,Th2 and Th17 cellular immunity compared with the control groups,and the immune response was mainly Th1 immune response.After 42 days of oral immunization with recombinant VP4/△HLJ-27,CD4⁺T,CD8⁺T and splenic lymphocyte proliferation were significantly higher than those in the control group,indicating that oral immunization with VP4/△HLJ-27 successfully activated the cellular immune response in mice.Piglets were orally immunized with recombinant VP4/△HLJ-27,and nasal and anal swabs and serum samples of immunized piglets were collected.Using PoRV virus as the coating antigen,the ELISA results showed that the specific Ig G antibody level in piglet serum and the specific SIg A antibody level in nasal and anal swabs were significantly higher than those in the control group,which same to the mice’s.Afterwards,the levels of cytokines IL-4,IL-2,IL-10,IL-12,IL-17 and IFN-γin the serum of piglets were significantly higher than those in the control group,indicating that the recombinant bacteria can stimulate piglets to produce Th1,Th2 and Th17 cell immunity,and the immune response was mainly Th1.In conclusion,oral administration of VP4/△HLJ-27 could induce mucosal,humoral and cellular immune responses in mice and piglets.