Identification Of Linear B Cell Epitopes Containing Peptides On The Hypervariable Region (1-496 Aa) Of Porcine Epidemic Diarrhea Virus S Protein

Porcine epidemic dirrhea?PED?is an acute,highly contagious porcine intestinal disease caused by Porcine epidemic dirrhea virus?PEDV?.The clinical symptoms are mainly diarrhea,vomiting,dehydration,anorexia and rapid weight loss,etc.Although pigs of various ages are susceptible to PEDV,piglets within 10 days are more susceptible to PEDV,and the mortality rate is as high as 100%.In 1971,the world's first PED outbreak occurred in shelf pigs and fattening pigs in England,and the symptoms were only manifested as diarrhea.In 1984,Xuanhua first confirmed the PEDV infection in pigs in some areas of China.Since the end of 2010,the outbreak of piglet diarrhea in pig farms in many areas from south to north and from east to west has been reported.And a new epidemic characteristics appeared,especially the high morbidity and mortality that resulted in the death of 1 million piglets.The mortality rate of piglets in one week is as high as 80%-90%,which has brought huge economic losses to the pig industry in China.The traditional vaccine originated from the classical strain of PEDV?CV777?does not provide good protection against the newly-appeared PEDV strain.At present,the mechanism for causing high pathogenicity of the new PEDV strain is still unclear.Antigen variability is one of the main reasons leading to the failure of the PEDV classical strain originated attenuated/inactivated vaccine to provide full protection against the epidemic strain.PEDV S protein plays an important role in the process of viral adsorption,invasion and membrane fusion of virus and infected host cells;S protein is also one of the main target proteins for inducing host to produce neutralizing antibodies.The amino acid sequence alignment S protein between the classical strain of porcine epidemic diarrhea virus and the epidemic strain showed that the S gene had undergone significant variation,and there was a hypervariable region(1-496 aa,S1_0A)at the N-terminus of the S protein.In order to explore the epitope change caused by gene variation in the hypervariable region of S protein and try to analyze the immune escape phenomenon of PEDV new epidemic strain at the epitope level,the following studies were mainly carried out in this study:1.Construction of expression vector of PEDV S1_0AThe recombinant vector carrying the codon-optimized PEDV vaccine strain?CV777?and the new epidemic PEDV strain?SD2014?S gene was used as a template to amplification of the PEDV classical strain S gene hypervariable region fragment(s1_0A^CV777)and the epidemic strain S gene hypervariable region fragment(s1_0A^SD2014)by PCR using the design specific primers.Then the amplified DNA fragment was inserted into pET-32a and two expression vectors pET-32a-S1_0A^CV777V777 and pET-32a-S1_0A^SD2014were constructed.The acquired recombinant expression plasmid was verified by sequencing and then transformed into E.coli BL21 competent cells for expression with induction by IPTG.The expression conditions,such as IPTG concentration and temperature were optimized.The preferred expression parameters were induction at37?with a IPTG concnentration of 0.8 mmol/L.The recombinant protein was expressed in the form of inclusion bodies.2.Preparation of polyclonal rabbit anti-PEDV S1_0AA serumThe recombinant protein was expressed in a enlarge volume under optimal conditions,then the cells were disrupted by ultrasonication and recombinant protein in forms of the inclusion body was collected by centrifugation.Subsequently,the recombinant PEDV S1_0AA was purified by cutting out the aimed band from SDS-PAGE gel and electroelution.In order prepare rabbit anti-PEDV S1_0AA sera,three New Zealand white rabbits were immunized with the purified PEDV S1_0A^CV777or PEDV S1_0A^SD2014?0.5 mg/mouse?.The rabbits were boosted 4 times every 14 days post the first administration.One rabbit was injected with PBS as negative control.The blood was taken from the ear veins on the 14th,28th,42th,and 56th after the first immunization,and the antibody level in the sera was detected by indirect ELISA.The rabbits were euthanized on the 58th day and the blood was collect from carotid artery.The antibody titers of rabbits were all higher than 1:1 638 400 which indicated that the polyclonal rabbit anti-serum obtained had a good sensitivity.Immunohistochemistry and immunofluorescenceanalysisshowedthattheobtainedanti-S1_0A^CV777and anti-S1_0A^SD2014polyclonal antibodies had a good specificity.3.Screening the linear B cell epitopes on PEDV S1_0A^CV777V777 and S1_0A^SD2014In order to construct the expression vector pXXGST-3-P1^P61,DNA fragemts encoding a serial of 16-mers which overlapped 8 aa eachother and covered the full length of the PEDV S1_0A^CV777V777 and S1_0A^SD2014D2014 were designed,synthesized and annealed in vitro.Then the dsDNA were inserted into the expression vector pXXGST-3.The recombinant plasmid with the correct sequence was transformed into E.coli BL21competent cells.Serial GST-fusion expressed 16-mers with an overlap of 8 aa residues covering the entire S1_0A^CV777V777 was produced in E.coli,and from which linear BCE containing 16-mers were identified by using western blot with prepared anti-S1_0A^CV777serum as the primary antibody.The conserved linear BCE containing 16-mers on S1_0A^CV777were identified with polyclonal serum against the counterpart of the S protein of PEDV SD2014(S1_0A^SD2014).A total of 34 positively reactive 16-mers were identified on PEDV S1_0A^CV777V777 using anti-PEDV S1_0A^CV777V777 polyclonal antiserum and anti-S1_0A^SD2014polyclonal antiserum.Among them,16 are epitope peptides recognized by two sera,12are epitope peptides specific for anti-PEDV S1_0A^CV777V777 serum,and 6 are epitope peptides specifically recognized by S1_0A^SD2014D2014 serum.Serial GST-fusion expressed 16-mers with an overlap of 8 aa residues covering the entire S1_0A^SD2014was produced in E.coli,and from which linear BCE containing 16-mers were identified by using western blot with prepared anti-S1_0A^SD2014D2014 serum as the primary antibody.The conserved linear BCE containing 16-mers on S1_0A^SD2014were identified with polyclonal serum against the counterpart of the S protein of PEDV CV777 vaccine strain(S1_0A^CV777).Using anti-S1_0A^SD2014D2014 polyclonal antibody serum and anti-S1_0A^CV777V777 polyclonal anti-serum,28positive reactive 16-mers on PEDV S1_0A^SD2014were identified,of which 13 16-mers could be recognized by both sera and 12 16-mers were specific for anti-PEDV S1_0A^SD2014D2014 serum.Meanwhile,3 16-mers were specifically recognized by anti-S1_0A^CV777serum.