Construction Of Lactobacillus Plantarum With Surface Displayed Porcine IFN-?3 And Its Functional Resistence To PEDV And TGEV In Vitro

Lactic acid bacteria as a probiotic gram-positive bacteria,can ferment carbohydrates.Lactobacillus plantarum(Lb.plantarum),characterized with high adhesion and strong hydrophobicity,can not only colonize in intestinal tract and become a dominant flora,thus exerting its immunoregulatory activity,but also directly capture viruses and play an antiviral role.As a probiotic,it is widely used in delivering drugs or protective antigens.Anchoring protein,as a surface display element,can anchor the heterologous protein to the surface of the new functional Lb.plantarum,which increases the local concentration of the heterologous protein and reduces its hydrolysis in the digestive tract.Porcine epidemic diarrhea virus(PEDV)and porcine transmissible gastroenteritis virus(TGEV)are two types of intestinal coronavirus that cause symptoms such as diarrhea,dehydration,and vomiting in piglets and have a high mortality rate.The co-infection of the two viruses is becoming increasingly frequent.Therefore,it is particularly important to develop a new drug to prevent and treat diarrhea in piglets infected with mixed viruses.Type III interferon(IFN-?)plays an important defense role in the early stage of anti-viral in epithelial tissue.IFN-? inhibits viral infection and replication through the Jak-STAT pathway,upregulating the expression of ISGs in downstream of the pathway,and brings adjacent healthy cells into an antiviral state.In order to construct new functional Lb.plantarum with surface displayed porcine IFN-?3,the antiviral activity of interferon was combined with the probiotic properties of lactic acid bacteria to exert the antiviral function of new functional lactic acid bacteria.The study is divided into four parts:(1)Firstly,the structure of CWA protein was predicted,and the anchor gene CWA was obtained to construct the anchoring expression new functional Lb.plantarum(Lb.plantarum-pSIP-409-EGFP-CWA).Flow cytometry,Western Blot,direct immunofluorescence and other assays were used to determine the optimal expression conditions,growth rate,and ability to display EGFP on the surface of new functional Lb.plantarum.(2)Secondly,the recombinant E-coli BL21-pET-30a-IFN-?3 was constructed,and the expression of the target protein was detected by Western Blot after induction by IPTG.The IFN-?3 protein was purified,renatured and the activity of the recombinant protein against PEDV and TGEV was verified.(3)Thirdly,new functional Lb.plantarum-409 IC and Lb.plantarum-409P?I were constructed and detected by flow cytometry and Western Blot which proved that the CWA anchoring method can more efficiently anchor porcine IFN-?3 than that in pgsA?.(4)The induced new functional bacteria and IPEC-J2 cells were co-cultured,and IPEC-J2 cells were infected with PEDV and TGEV respectively,and confirmed by real-time quantitative PCR(qPCR).Two new functional strains Lb.plantarum-409 IC and Lb.plantarum-409P?I can significantly inhibit the RNA transcription level of PEDV and TGEV in IPEC-J2 at different concentrations and time points(0.01<p<0.05),especially the anti-viral effect of new functional strain Lb.plantarum-409 IC seemed stronger.By indirect immunofluorescence,the viral load of the new functional strain treatment group was significantly reduced in the IPEC-J2/PEDV system and the IPEC-J2/TGEV system compared with that in the control group.IPEC-J2 cells stimulated by new functional Lb.plantarum-409 IC and determined by qPCR assay,interferon-stimulated gene: ISG15,Mx1,IFITM3 were significantly up-regulated.This result indicates that the new functional strain can inhibit the replication of the virus by increasing the relative expression of antiviral gene in IPEC-J2.This study not only confirmed that both CWA and pgsA? can successfully display porcine IFN-?3 protein on the surface of Lb.plantarum,but also found that the new functional strain Lb.plantarum-409 IC is more effective suppression of PEDV and TGEV replication.This study laid a foundation for the prevention and treatment of PEDV and TGEV infection in pigs with new lactic acid bacteria preparations.