Preparation Of Monoclonal Antibody Against S1-1Protein Of PEDV And Construction Of The Surface Display Vector Of Saccbaromyces Cerevisiae Inducted By Glucose

Porcine Epidemic Diarrhea(PED) was reported for the first time in Britain andBelgium in1978and characterized by diarrhea, vomit and dehydration. The pathogenicvirus(PEDV) belongs to Nidovirales, Coronaviridae, Coronavirus. Porcine epidemicdiarrhea virus is positive-strand RNA genome of approximately28kb. It has six openreading frames are contained in the genome of PEDV. The spike(S) protein is one of thestructural protein and encoded by ORF2. It has great significance for diagnosis andprevention of PED. In this study, according to the Genbank, we cloned and expressed partsof the S1-1protein gene. The BALB/c mice were immunized with the purified His-S1-1fusion protein. The monoclonal antibody against S1-1protein of PEDV was developed. Inaddition, in order to reduce the cost of inducible expression, we modified the eukaryoticexpressing plasmid of s.cerevisiae pYD1-S1-1.1. Cloning and prokaryotic expression of S1-1protein gene of PEDVAccording to the gene sequence of the spike(S) protein which was published inGenBank, one special pair of primers were designed to amplify the S1-1protein genefragment by RT-PCR. The production is420bp which is named S1-1. It was cloned into theexpression plasmids pET-32a(+) and pGEX-4T-1. The recombinant expression plasmidswere transformed into the expression system BL21(DE3)plysS and inducted by IPTG. TheresμLt of SDS-PAGE indicates that two fusion proteins were expressed in a high level. Forthe highest expression, The optimal induction conditions of two fusion protein wereexplored in our test. The best concentration of IPTG of His-S1-1is at0.6mmol/L,and theother one is at0.4mmol/L. His-S1-1is highly expressed at37℃for4h, at the sametime,.GST-S1-1is highly expressed at37℃for5h. The fusion proteins were purified byMagneGST and HisTrapTMFF protein purification kit. In our experiment, the concentrationof purified fusion protein His-S1-1is at2.6mg/mL, while GST-S1-1is at1.1mg/mL.Theindirect ELISA assay indicated that two recombinant proteins have a greatreactionogenicity.2. Development of monoclonal antibodies against S1-1protein of PEDVThe BALB/c mice were immunized by purified protein His-S1-1for four times. Wedetected the titer of serum antibody of immunized mice by indirect ELISA. The hightesttiter of serum antibody is at1:5.12×10~5when we used the purified protein His-S1-1asdetecting antigen, while GST-S1-1is at1:1.28×10~5. Spleen cells from immunized micewere fused with SP2/0myeloma cells. We used the detecting antigen of purified proteinGST-S1-1to screen hybridoma cells by indirect ELISA. Positive hybridoma cells weresubcloned by limiting dilution method. Finally we obtained one positive clone. The titer ofpurified ascites was at1:2.56×10~5; Western blot analysis showed the monoclonalantibodies against S1-1protein coμLd specifically combine with the fusion proteins His-S1-1and GST-S1-1.3. The shuttle plasmid pYD-S1-1of saccharomyces cerevisiae was modified.According to the gene sequence of glycerol3-phosphate dehydrogenase published inGenBank, one special pair primers were designed to amplify the glycerol3-phosphatedehydrogenase gene fragment by PCR. The gene fragment is713bp. Finally, the originalgalactokinase (GAL1) weak promoter was replaced of3-phosphate dehydrogenase strongpromoter (GPD1) by homologous recombination.