Recombinant Vaccine Of Goatpox Virus Vectored Antigen Genes P12A3C Of Foot And Mouth Disease Virus Construction And Evaluation Of Immune Effect

Foot and mouth disease(FMD)and Capripox(CP),caused by foot and mouth disease virus(FMDV)and Capripoxvirus(CPV)infections,respectively,are included in the list of animal diseases notifiable to the World Organization for Animal Health(OIE).FMD and CP are highly contagious viral disease that affects the sheep industry in China,including international trade in livestock and their products.Nowadays,vaccination is still the only effective way to prevent and control the spread of the FMDV and CPV infection.Among them,the inactivated vaccine is used to against FMD and the live attenuated vaccine is immunized against sheep pox.However,the prevention and control effect is affected due to the interference in two vaccines each other and the cumbersome immunization procedure.Therefore,it is urgently need to develop new vaccines with the immunization strategy of one-shot multi-prevention against CP and FMD.Live attenuated virus vector vaccine is an advantageous tool for the development of multivalent vaccines.In this study,sheep pox virus(SPV)was used as a viral vector to construct a transfer vector and the antigen gene P12A3 C of FMDV was recombined into the genome of SPV through homologous recombination method.The recombinant GPV containing the FMD antigen structural protein gene was rescued and the immune effect was further evaluated in goats.These findings will provide data support for the study of live vector vaccines against both FMD and SPV.The main research advances are as follows:1.Construction and identification of the recombinant GPV containing the FMD antigen gene of the P12A3 C geneThe shuttle plasmid p VAX1-TK-P containing the TK gene homologous arm and Vaccinia virus double promoter was constructed for the recombination of Goatpox virus.Another shuttle plasmid p TK-P-GIg containing screening genes was also constructed,which carried both pressure screening gene GPT and marker screening gene GFP.The antigen gene P12A3 C fragment of type O FMDV was inserted into the polyclonal site of shuttle vector p TK-P-GIg to obtain the p TK-P-GIg-P12A3 C plasmid,which was used for homologous recombination with GPV.In order to verify the function of the promoter in the shuttle plasmid PVAX1-TK-P,the p TK-P-GFP plasmid was constructed by inserting GFP gene downstream of the P7.5promoter.p TK-P-GIg,p TK-P-GIg-P12A3 C and p TK-P-GFP were transfected into primary cells of the lambs testis(LT)cell,respectively.The results showed that green fluorescence could be observed in cells transfected with each plasmid,and green fluorescence appeared earlier in cells with p TK-P-GFP plasmid transfection,because its promoter p7.5 is the early promoter of Vaccinia virus,which was consistent with expectations.Cell proteins transfected with PTK-P-GIg-P12A3 C were harvested and analyzed by Western blotting.The results showed that cell lysates could react specifically with FMDV VP1 antibody.This suggested that the constructed shuttle plasmid p TK-P-GIg-P12A3 C containing TK gene and screening gene and FMDV antigen gene controlled by double promoter could be used for the construction of recombinant GPV.The recombinant plasmid PTK-P-GIg-P12A3 C containing FMDV antigen gene was transfected into GPV-infected LT cell.The p TK-P-GIg-P12A3 C plasmid recombined homologous with the GPV virus.The recombinant virus was screened by marker gene and resistance gene and purified by limited dilution method.The recombinant virus was cultured in large quantities,and then the virus genome was extracted.The purity and exogenous genes of the recombinant virus were identified by amplifying TK gene and P12A3 C gene.The results showed that only one band of TK gene was amplified,and a fragment corresponding to the size of the target band was amplified by P12A3 C gene primer.Sequencing analysis of the amplified target gene showed that the sequence was the same as the insertion P12A3 C sequence.The results showed that the recombinant r GPV/FMDVP12A3 C was successfully obtained.Meanwhile,marker genes and antigen genes were amplified by RT-PCR to detect the transcription of recombinant virus.The results showed that all the inserted genes,including marker gene GFP,resistance gene GPT,IRES sequence and FMDV antigen gene P12A3 C,could be detected at RNA level.The P12A3 C gene expression of recombinant virus was detected by indirect immunofluorescence assay and Western blotting.The results showed that the FMD antigen gene was expressed normally and the expressed protein had good reactivity.2.Evaluation of immune effect of the recombinant GPV containing the FMD antigen gene of the P12A3 C geneGoats were immunized with r GPV/FMDVP12A3 C recombinant virus,commercial goat pox vaccine positive control group,O-type FMD inactivated vaccine positive control group and PBS negative control group,and then the antibody response were detected.The results showed that the r GPV/FMDVP12A3 C recombinant virus could induce a higher level of anti-GPV antibody,and the antibody titer was similar to that of the commercial goat pox vaccine group,but the level of the induced FMD antibody titer was not higher than that of the O-type FMD inactivated vaccine.Although the titer of FMDV antibody increased after the second immunization of r GPV/FMDVP12A3 C,the FMDV antibody level was significantly lower than that induced by O-type FMDV inactivated vaccine.Enhancing the immune response by increasing the immune dose and using adjuvants and other methods may improve and enhance the immune efficacy of the recombinant virus exogenous genes.In conclusion,this study lays the foundation for the immunization strategy of one-shot multi-prevention of FMD and CP,and also provides a reference for the development of live vector vaccine of sheep pox recombinant with other exogenous genes.