| Porcine Epidemic Diarrhea?PED?is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus?PEDV?.In recent years,the disease has presented new epidemic characteristics,such as extremely high mortality rate?90-100%?and younger age of sick piglets.Pigs of all ages are susceptible,but suckling piglets are more severely infected.A large epidemic of PED has been spreading worldwide since 2010,which lead to poor vaccination.It is urgent to develop a new generation of PEDV vaccine for new epidemic strains,considering the changes of immunogenicity and virulence caused by the gene variation under long-term immune pressure.By the method of reverse genetic manipulation based on RNA recombination,a recombinant PEDV?named rPEDV-D375F?was constructed in our lab by substituting S gene of the DR13 vaccine strain with that from a prevalent strain.On this basis,this study took recombinant PEDV laboratory culture technology as a breakthrough point,preliminary evaluated vaccine immunization effectiveness,which could lay a good technical foundation for the development of recombinant rPEDV-D357F inactivated vaccine.The main research contents and results are summarized as follows:1)Establishment and test of Vero cell bank for production.In this paper,cell bank forPEDV vaccine production was established by passaging Vero cells from generation 1st to generation 100th.These cells were tested for cytology,tumorigenicity,foreign virus,mycoplasma and asepsis.The results showed that Vero cells were free of contamination and met the basic requirements of vaccine production cells and could be used as Vero cell bank forPEDV production.2)Establishment of lab culture protocol for rPEDV-D375F.To obtain the high and stable titer of recombinant strain in this study,the culture protocol of recombinant PEDV was established by optimizing the optimal infection complex number?MOI?,infection time,culture temperature and drug collection method mode of the virus.The results showed that the optimum culturing condition was 0.01 infection complex number MOI,18-20 h the optimal infection time,37°C the optimum culture temperature,and repeated freezing and thawing 3 times before drug collection.The process can ensure the titer of the recombinant strain stably above106.5TU/m L.3)Preparation and immunogenicity evaluation of rPEDV-D375F inactivated vaccine.In this paper,the inactivated conditions of virus was explored firstly,and optimal conditions were that the rPEDV-D375F strain was inactivated with 0.2%formaldehyde at 37°C for 24 h.Then,inactivation vaccine was prepared using inactivated virus along with 206 adjuvant and test from physical character,aseptic and safety.Finally the inactivated vaccine with a virus titration of 1×106.5TU/m L before inactivation were used for immunization test on PEDV negative Landrace,which were divided into three groups,a mock group and two immunization groups receiving two time intramuscular?IM?injections of 4 m L/head and 8 m L/head each time,respectively,at a 14 d interval.Blood and serum were collected and isolated respectively before and after immunization and specific antibodies were detected.The results showed that specific anti-PEDV antibody could be detected 14 d post immunization?d.p.i?in the 4 m L dosage group.The antibody could be detected for 49days and reached a top level at 21 d.p.i..Anti-PEDV antibody could be detected on7th d.p.i.until 77th d.p.i.in the 8 m L dosage group,reaching a peak level at 21 d.p.i.Therefore the results suggested that the inactivated recombinant PEDV vaccine has good immunogenicity.In conclusion,the laboratory culture technology of Vero cell bank forPEDV production and recombinant strains with stable titer of 1×106.5TU/m L or more were established,respectively.The inactivated vaccine of recombinant PEDV was prepared and proved to possess a good immunogenicity by porcine immunoassay in vivo.This study provide a solid technical foundation for developing inactivated recombinant PEDV vaccine. |
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