The Construction And Immunogenicity Evaluation Of Recombinant Porcione Adenovirus Vector Expressing PEDV S Protein Core Antigen Region

Porcine Epidemic Diarrhea(PED),an acute,highly contagious intestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV).Its mortality rate can reach80%-100%.The disease has now become one of the most intractable problems affecting the world’s pig industry.Since 2010,PEDV mutants have started to break out in pig farms in most parts of China,and the traditional PED vaccine has significantly reduced the prevalence of epidemic.The S protein is the most important protective antigen of PEDV and the largest variant protein.Based on the evolution analysis of S protein,most of the current prevalence viruses are gene type II.While the traditional vaccines are gene type I and gene type II epidemic and gene type I traditional vaccine poisoning in S gene,especially the core antigenic region of S1(COE)has been mutated in large numbers and has caused changes in S protein results and partial glycosylation changes.In order to solve the poor protection of traditional PED vaccines and to develop new vaccines with high cross protection,this study initially performed high frequency site mutation and glycosylation site modification of S gene COE(removing glycosylation sites or adding glycosylation).Sites,followed by wt COE(COE)and mutant COE(m COEs)were cloned into porcine adenovirus type 3(PAd V-3)respectively,and recombinant adenoviruses PAd V3-COE and PAd V3-m COEs were constructed.Finally,mice were immunized with recombinant adenovirus PAd V3-COE and PAd V3-m COEs to screen for S gene COE mutants that can induce high levels of neutralizing antibodies and the basis for the development of a new generation of high-efficiency PED adenoviral vector oral vaccine.This study consists of two parts:1.Construction and identification of recombinant PAV-3In this study,we analyzed the high frequency mutations and glycosylation mutation sites of the CO protein region(491-638-aa)of different S strains and introduced glycosylation with reference to the MERS related literature.The COE-mutated nucleic acid sequence with the homology arm was amplified by PCR and fusion PCR and the engineered mutant fragment was inserted into the PAd V3 genome by In-fusion recombination technique to construct a recombinant porcine adenovirus vector p FPAV3-COEs.PAd V3-COE and PAd V3-m COEs linearized with Pac I were transfected into VR1 BL cells by Lipofect reagent,and the recombinant viruses r PAd V3-COE and r PAd V3-m COEs were packaged.After virus passage,CPE detection,PCR and Western blot were used for recombinant virus construction and protein expression,respectively.2.Neutralizing antibody detection of recombinant PAV-3Mice were immunized subcutaneously with rPAd V3-COE and rPAd V3-mCOEs,and serum-inducing levels were detected at 14 days after the second immunization and 14 days after the third immunization,and COE mutants capable of inducing highly neutralizing antibodies were screened.The results of detection of neutralizing antibodies showed that half of the mice neutralized antibody titers were less than 1:16 after immunization with wild-type COE-recombinant virus,and the high-frequency mutant recombinant virus More than half of the mice after immunization of mice with PAV3-KB3-B2-530 L,PAV3-KB3-B2-536 V,PAV3-KB3-B2-558 T,PAV3-KB3-B2-572 N,PAV3-KB3-B2-614 A,PAV3-KB3-B2-639 K,exceeded 1:16,glycosylation-removed recombinant virus PAV3-KB3-B2-N55 A,glycosylation into the recombinant virus PAV3-KB3-B2-563 N and PAV3-KB3-B2-621 N more than half of the mouse neutralizing antibody titer is greater than 1 :16.