Expression And Identification Of B Cell Epitope-embedded S-layer Protein Of PEDV S2 Gene In Lactobacillus Paracasei

Porcine epidemic diarrhea(PED)is a highly contagious infectious disease caused by porcine epidemic diarrhea virus(PEDV).The disease was first identified in the UK in the1971 and reported in Asia in 1982.PEDV had spread to many African and European countries causing high piglet mortality up to 80-100%.The absence of PEDV mutation caused a huge threat to the global pig industry。 because traditional inactivated vaccines and attenuated vaccines had low serum antibody titers against the PEDV mutant strains especially after the emergence of highly pathogenic variant in 2010.With the emergence of highly pathogenic variant strains in 2010,these traditional PEDV inactivated vaccines and attenuated vaccines usually have low serum antibody titers against the newly emerged PEDV variant strains and can no longer provide effective protection.Therefore,the research on new vaccines has become a hot research topic in recent years.This study attempts to design a new vaccine to meet the needs of PED prevention and control.In this experiment,biological software and online database were used to screen out the B cell epitope of PEDV S2 gene,and the epitope peptide was synthesized artificially.This epitope peptide was conjugated with keyhole limpet hemocyanin(KLH)as an antigen to immunize 6～8 week-old female BALB/c mice.First,The fusion gene SLP-Epitope S2 was amplified and cloned into the lactic acid bacteria expression vector p TRK892 to construct the recombinant plasmid p TRK-SLP-Epitope S2.After confirming that the target gene was accurately connected to the vector by PCR,double enzyme digestion and gene sequencing,the recombinant plasmid was electrotransformed into Lactobacillus paracasei,and positive clones were screened by PCR.The positive clones were picked for non-induced expression,and the expression products were identified by SDS-PAGE,Western blot,immunofluorescence and Li Cl treatment.The test results are as follows:1.Screen out the B cell epitope peptide through biological software and online database,namely: MQYVYTPTYYML.After immunizing with the polypeptide,the antibody titer of the mouse serum before fusion reached 1:2000.The ELISA results of hybridoma cell culture supernatant and ascites showed that the antibody titer reached 1:4000.2.The results of PCR,double-enzyme digestion and sequencing were consistent with the theoretical values,which proved that the recombinant plasmid p TRK-SLP-Epitope S2 was successfully constructed.The fusion gene SLP-Epitope S2 was successfully expressed in the lactic acid bacteria expression system,and the target band appeared at about 48 k Da in SDS-PAGE and western blot results,which was consistent with the theoretical value.3.The results of immunofluorescence detection showed that most of the recombinant Lactobacillus paracasei were excited with obvious green fluorescence,while the control group had no green fluorescence.The results of SDS-PAGE and Western blot of recombinant Lactobacillus paracasei and control bacteria treated with Li Cl showed that the recombinant Lactobacillus paracasei had a target band at a size of 48 k Da,while the control bacteria had no target band,suggesting that the recombinant fusion protein expressed Surface with Lactobacillus paracasei.Conclusion: This study laid the foundation for the study of Lactobacillus live bacteria carrier epitope peptide vaccine.