Construction Of B-cell Epitope-embedded S-layer Protein Carrier Of PEDV S2 Gene And Its Antigenicity Analysis

Porcine Epidemic Diarrhea Virus(PEDV)is classified to coronavirus type I and causes severe diarrhea in pigs.The main character of this disease is acute watery diarrhea,dehydration and vomiting.High death rates caused by PEDV in piglets,which bring huge economic losses to the pig industry.In recent years,the rapid development of molecular biology techniques leads to many breakthroughs on the genetic structure,gene function,pathogenic molecular mechanism of PEDV,even the virus detection and vaccine development.However,we still need a deeper understanding for the overall control of PEDV.The main points and results are as follows:1.B cell epitope gene of S2 gene of PEDV was screened and obtained by biological software and online database.Then,it was inserted into the gene of S-layer protein of Lactobacillus acidophilus by Overlap PCR and the fusion gene SLP-EpitopeS2 was obtained.Finally,the fusion gene SLP-EpitopeS2 was introduced into the prokaryotic expression vector pGEX-4T-3 and the recombined pGEX-SLP-EpitopeS2 was successfully constructed.2.The constructed pGEX-SLP-EpitopeS2 was identified by PCR,restriction enzyme digestion and nucleotide sequencing,Then,it was induced to express in E.coli by IPTG.The expressed fusion protein SLP-EpitopeS2 was purified and detected by SDS-PAGE and Western blotting.The SDS-PAGE showed that the molecular weight of the fusion potein and GST-tag 77 KDa and 26 KDa,respectively.The results of the was Western blotting showed the fusion portein could be recognized by GST serum,and B cell epitopes were recognized by epitope-specific murine monoclonal antibodies(McAb)and epitope synthetic peptide,respectively.3.The fusion protein was further purified by gel-cutting purification and injected to immunize 6 weeks old BALB/c mice.The levels of total IgG,TgGl,and IgG2a of each group of mice were detected by an indirect ELISA method.The results showed that serum IgG levels after the third immunization were significantly higher than the previous two;the IgG levels in the experimental group were also significantly higher in the GST-tagged protein group and the PBS-immunized group(P<0.01).Subsequently,the IgG2a and IgGl levels of the mouse IgG subtypes showed that the levels of IgG2a and IgGl in the fusion protein immunized group were significantly higher than the GST-tagged protein group and the PBS-immunized group(P<0.01).The level of IgGl in the embedded protein GST-SLP-EpitopeS2 immunized group was significantly higher than that of IgG2a(P<0.05).This result suggests that the embedded protein GST-SLP-EpitopeS2 may be more prone to trigger the humoral immune responseThe results of this experiment further laid the foundation for the experimental study on the antigenicity and immunoprotection of the B cell epitope-embedded protein SLP-EpitopeS2 of the PEDV S2 gene.