Effctiveness Of Swine GM-CSF For Classical Swine Fever E2 Subunit Vaccine And Evaluation Of Immune Effectiveness Of PCV2-PPV Bivalent Subunit Vaccine

Classical swine fever is an acute or chronic,hot and highly contagious disease caused by the classical swine fever virus.Classical swine fever is of worldwide distribution,of great harm and a serious threat to the development of the pig industry.In our country long-term use of live attenuated vaccine of C-strain obtained good results and inhibited the epidemic and harm of classical swine fever in some extent.But the use of the C-strain live attenuated vaccine could not effectively identify on wild virus infections and it was not conducive for swine classical fever to purify and eliminate.Therefore,the study of new marker swine fever vaccine is imperative.In addition,porcine circovirus type 2（PCV2）and porcine parvovirus（PPV）are two important pathogens which can harm the healthy development of the pig industry.Mixed infection of PCV2 and PPV significantly increases the incidence of post-weaning multisystemic wasting syndrome（PMWS）and aggravates the reproductive disorders of sows,especially gilts.Therefore,the development of a safe and efficient bivalent vaccine has important significances for prevention and control of two diseases.Therefore,on the basis of developed swine fever E2 subunit vaccine in our laboratory,a baculovirus rBac-3GM-CSF which expressed GM-CSF protein was constructed.Finally the adjuvant effect of GM-CSF to E2 subnit vaccine was evaluated in rabbits to study a safer and more efficient new type swine vaccine.In addition,the systematic evaluation of ORF2+VP2 bivalent subunit vaccine was conducted in pigs.Details are as follows:1.Construction of recombinant baculovirus rBac-3GMCSF and preparation of subunit vaccineGM-CSF was amplified by PCR with the saved plasmid pFBD-GMCSF as template.And it was inserted into the baculovirus transferred vector pFBD-GMCSF to obtain the recombined plasmid pFBD-3GMCSF.The pFBD-3GMCSF was transformed into E.coli DH10Bac competent cells.The recombined pFBD-3GMCSF was attained by extracting from the purified blue white screenings.After it was indentified correctly by PCR,it was transfected into Sf9 cells to gain F0 generation of rBacmid-3GMCSF.The expression product was indentified by Western-blot and indirect immunofluorescence assay,the results indicated that GM-CSF protein was successfully expressed.The rBac-3GMCSF and rBac-3E2 were inoculated with the suspending Sf9 cells in 1.0 MOI to express GM-CSF and E2 protein.And two cell suspensions mixed with adjuvant（ISA 201VG）were emulsified at the mass ratio of 1:1 to prepare E2+GM-CSF subunit vaccine,target E2 protein content 20ug/ml and target GM-CSF protein content 5ug/ml respectively.Also E2 subunit vaccine was prepared,E2 content 20ug/ml.Finally,the quality control tests of vaccine（stability test,viscosity test and sterility test）were performed,the results showed no bacterial contamination of the vaccine and the physical properties of indicators were in line with the standard of vaccine emulsion.2.The study of the effect of porcine GM-CSF protein to swine fever E2 subunit vaccine2.1 Immunogenicity experiment in rabbitsThe prepared E2+GM-CSF complex vaccine and E2 vaccine which were produced by ISA201VG adjuvant were immuned with 2-months-old Japanese long-eared rabbits,C-strain live attenuated vaccine as the positive control group,PBS as the negative control group,intensive immunization was conducted 21 days after the first immunization,swine fever virus attack protection test was performed 21 days after the second immunization.Serologic test results demonstrated:after intensive immunization,the level of swine fever specific antibody the subunit vaccine group reached peak 21 days after the second immunization.And the average block rate reached 65%.It was of no significant difference among C-strain group,E2 group and E2+GM-CSF group（P>0.05）.But the block rate of each vaccine group was much higher than that of PBS negative group（P<0.05）.The results showed subunit vaccine could induce high level of the specific E2 antibody and achive good immune effect.Meanwhile,the result of the peripheral blood lymphocyte proliferation experiment was in accordance with that of serum IFN-γcontent determination.The stimulation index value and IFN-γcontent were both higher than PBS negative control group（P<0.05）,it indicated that the subunit vaccine was capable of inducing an effective immune responses in the body.The results of determination of IL-4 content showed the IL-4 content of E2+GM-CSF group raised but not significantly（P>0.05）,however,that of E2 group upregulated significantly（P<0.05）.2.2 The challenge of Classical swine fever virus56 days after the first immunization,100 RID₅₀of C-strain were inoculated by each rabbit.The body temperature of every rabbit was monitored during the next 144h.Results showed that the body temperature of rabbits from PBS negative group rose,lasting for24h.It was the typical stereotypes thermal reaction.And the body temperatures of rabbits immuned with E2+GM-CSF and E2 were normal.14 days after challenge,the rabbits’spleens were collected and the CSFV copy numbers in spleen were detected by real-time quantitative PCR method.The results also implied that the CSFV copy numbers of vaccine group were significantly lower than that of the negative control group（P<0.05）.The swine fever virus copy numbers of E2+GM-CSF group were much lower than that of E2 group,and the difference was significant（P<0.05）.These results above indicated that the subunit vaccine could provide 100%effective protection against CSFV.3.The evaluation of immunogenicity of PCV2-PPV（ORF2+VP）bivalent subunit vaccine in pigsRecombinant baculovirus rBac-3ORF2 and rBac-3VP2 saved in the laboratory was respectively inoculated in Sf9 cells with 1.0 MOI,cell suspensions were collected to get ORF2 protein and VP2 protein.And two proteins were mixed with Seppic Gel02 adjuvant to emulsify.Then two scales of PCV2-PPV bivalent subunit vaccine were produced,50ug ORF2+80ug VP2 and 50ug ORF2+100ug VP2.Storage tests were conducted at 0,3,6months after vaccine saved.Results indicated that no bacterial contamination of the vaccine and the physical properties of indicators were in line with the standard of vaccine emulsion.The immune efficacy of PCV2-PPV（ORF2+VP2）bivalent subunit vaccine was evaluated in 21-day piglet.Commerial PPV inactivated vaccine and PCV2 subunit vaccine were used as control.A booster immunization was conducted 21d post first immunization.Serum antibody was detected on 0d,21d,42d and 63d.The results of serological test showed that the level of anti-PCV2 antibody of the vaccine group was higher than that of the commercial PPV vaccine 42d after first immunization（P<0.01）.The level of bivalent subunit vaccine was higher than that of commercial inactivated PPV vaccine,but it was of no difference（P>0.05）.Meanwhile the level of anti-PPV antibody of the ORF2+VP2（50+80）vaccine group was higher than that of the commercial PCV2 vaccine（P<0.05）.And the level of commercial inactivated PPV vaccine was highest,and it was of significant difference to negative control group（P<0.01）.However,the level of ORF2+VP2（50+100）was of no difference to negative control group.These results show that the bivalent subunit vaccine could stimulate the body to produce higher levels of humoral immunity of PCV2 antibody,also could induce anti-PPV antibodies.And the VP2 protein content of subunit vaccines was not positively colrrelated with PPV antibody level.Hemagglutination inhibition test results demonstrated that the HI level of subunit vaccine group was nearly equivalent to that of commercial PPV vaccine group,the difference was not significant（P>0.05）.And it was higher than the commercial PCV2 vaccine group,the difference was significant（P<0.001）.