Screen Out The Major Epidemic Strains Of Porcine Epidemic Diarrhea And Construction Recombinant Pseudorabies Virus Expressing S Protein

Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),is an acute and highly contagious intestinal disease in swine.The major clinical symptoms of this disease are diarrhea,vomit and dehydration.Since late 2010,explosive outbreaks of PED in China has killed a huge number of piglets.Hence,PED is one of the most significant diarrhea disease in swine,frustrating challenge to the pig industry in China.The type of PEDV prevailing in China belongs to type 2,the differences with British CV777 are in S gene with base insertion or deficiency.Which having only 85% homology which belongs to type 1.The traditional PED vaccine strains belong to type 1,with poor protective effect in pigs.So far,there is no ideal effective vaccine to prevent or control the emergence of PED.On the other hand,Pseudorabies Virus(PRV)has a huge genome,which is nearly150 Kb.It has several characteristics,such as broad range of host,human nonsusceptible,,containing many non-essential genes,and stable for recombination.In latest years,it has become to be a hot topic in the field of genetic engineering vaccine that PRV works as a vector for various exogenous proteins to reform a recombinant virus,where PRV-bac is the most prominent one.There has important practical significance which construction recombinant of Pseudorabies Virus Expressing S Protein.By sequencing and analysis S and ORF3 genes we separated twelve strains infected with PEDV from 2011-2016 of Zhejiang province,the results showed,The twelve strains all belong to the Ⅰgroup,share high sequence homology 97.8%-99.9%with China seperated strains(BJ-2011 and Hu N)in Genbank published,while they have low sequence homology 93.3%-94.9% with main vaccine strains(civil vaccine strain CV777 and Korean attenuated strain DR13).The twelve epidemic strains ORF3 gene amino acids sequence homology 96.9%-100% between each other.The Sgenetic amino acid sequence homology was 100% between NB120929 、 FY140714 and HZ150314,can choose any one as a epidemic strain.And 95.6%-96.4% homology with vaccine strain CV777,97.8%-99.6% homology with civil separated BJ-2011,96.9%-99.6% homology with Korean strains Chinju99 and DR13.By analysis Zhijiang PEDV S and ORF3 genes genetic evolutionary from 2011-2016.The main reason is S gene mutation.By analysis S and ORF3 genes genetic evolutionary relationships,found out the major epidemic strains(ZJ131016)of PEDV in Zhejiang.and optimized the S1 and S2 genes in order to suitable expression in eukaryotic system,after inserted optimizated S1 and S2 genes into pPRV-Bac-EF1 by Red/ET two-step method,structured mutant pPRV-S1-ΔgIE and pPRV-S2-ΔgIE which lack of g I and g E genes by calcium phosphate precipitation method to obtain infective recombinant viruses r PRV-S1-ΔgIE and r PRV-S2-ΔgIE.and delete r PRV-S1-ΔgIE and r PRV-S2-ΔgIE Kan gene by L-arabinose induce,obtain infective recombinant viruses r PRV-S1-ΔgIE and r PRVS2-ΔgIE.Then the two plasmids were transfected into BHK-21 cells The expression of S1 and S2 proteins were confirmed by western blot.Results showed that only rPRV-S1-ΔgIE expressed S1 protein has good immunogenicity by immunized BALB/c mice,but r PRV-S2-ΔgIE expressed S2 protein has poor immunogenicity.Laid a foundation for the further development of PRV and PEDV bivalent recombinant live vaccines.