| Classical swine fever（CSF）is an economically important highly contagious disease in the pig industry,with classical swine fever virus（CSFV）as its pathogen.CSF outbreaks usually cause enormous economic losses,and CSF has been classified as a class A infectious disease by the World Organization for Animal Health（OIE）.Currently,CSF outbreaks are still reported in most countries of Central America,South America,and Asia.In recent years,epidemic patterns of CSF have changed greatly,which are mainly sporadic with no typical clinical symptoms.Recent research results have shown that epidemic strains in the CSFV gene group II play leading roles in the prevalence of CSF worldwide.In China,current epidemic strains of CSFV can be divided into two gene groups and four gene subgroups（subgroups 1.1,2.1,2.2,and 2.3）.The genes of epidemic strains of CSFV are gradually deviating from the vaccine strains because of the strengthened immunological pressure after long-term use in pigs.Although live attenuated vaccine of C-strain has good immunogenicity and safety,which has played an important role in the prevention and control of CSF.However,duo to the implementation of CSF eradicative plan,the development of CSF marker vaccine inducing rapid protection combined with the possibility to differentiate vaccinated animals from those infected with CSFV becomes more and more important.In addition,CSF and porcine circovirus 2b（Porcine circovirus type 2b,PCV2b）both can cause failure in propagation of pigs.The Cap protein of PCV2 can be independently assembled into virus-like particles（VLPs）,which has potentialas antigen vehicle carrying foreign epitopes.In this study,the main immunogenic E2 gene of CSFV of different genotypes was amplified and synthesized.E2 proteins from different CSFV genotypes were expressed in a baculovirus expression system,and new subunit vaccines against CSF were obtained.The immunogenicity of these vaccines in pigs and rabbits was studied.In addition,virus-like particles（VLPs）of chimeric porcine circovirus type 2（PCV2）were generated by replacing the nuclear localization signal（NLS）of PCV2 capsid protein（Cap）with CSFV T-cell epitope,CSFV B-cell epitope,and CSFV T-cell epitope conjugated with B-cell epitope.Immunogenicities of the chimeric VLP vaccines were evaluated in mice and rabbit.And furthermore,transgenic pigs with overexpressed sIFITM3 were constructed in our laboratory,and anti-FMDV was evaluated in the transgenic pigs.The contents as following:1.Construction and immunogenicity study of subunit vaccines with glycoprotein E2 expression of genotypes 1.1 and 2.1 of CSFVGenes of 1.1E2 and 2.1E2 fused with the signal peptide fragment of honeybee melittin signal peptide at the N-terminal were obtained by overlapping PCR.These genes were then inserted into the recombinant baculovirus transfer vector p FBD.The recombinant transfer plasmids p FBD-3-1.1E2 and p FBD-3-2.1E2 were constructed,each of which carried three copies of the target gene.The recombinant plasmids were transformed into Escherichia coli DH10Bac,and positive colonies were screened and purified by continuous blue white screening.After extraction,the bacmids were transfected into Sf9 insect cells by liposome Cellfectin?II reagent,and recombinant baculoviruses Ac-3-1.1E2 and Ac-3-2.1E2 were obtained.Indirect immunofluorescence and Western blot experiments showed that the proteins of 1.1E2 and 2.1E2 were successfully expressed by recombinant baculoviruses Ac-3-1.1E2 and Ac-3-2.1E2 in insect cells,respectively.Both proteins showed good immunological activities and response to anti-E2 monoclonal antibody.However,these proteins were expressed intracellularly,without extracellular secretion.With the addition of equal volumes of adjuvant 206,doses of 40μg of target proteins of 1.1E2 and 2.1E2 were emulsified as subunit vaccines,namely,1.1E2,2.1E2,and 1.1+2.1E2.Animal experiments demonstrated that all three subunit vaccines could induce anti-CSFV antibodies and neutralizing antibodies in both rabbits and pigs.Moreover,all the vaccines could induce specific lymphocyte proliferation.The stimulation index（SI）of the C-strain was slightly higher than that of groups 1.1E2,2.1E2,and 1.1+2.1E2,with no significant differences（P>0.05）.The results of cytokine detection were similar to that of lymphocyte proliferation,and the immunological response in C-strain was higher than that in other groups.However,compared with the negative control（PBS）,all the tested groups showed significant differences.Rabbit immunization experiments showed that the body temperature of the tested rabbits slightly increased in the 1.1E2 and 1.1+2.1E2 groups after challenge,but no fever reactions were noted.In group 2.1,two of the immunized rabbits showed fever reactions of CSF.In pig immunization experiments,all the animals in the 2.1 group showed increased body temperature and typical symptoms of CSF,and all died within a few days after challenge.The body temperature of 3 tested pigs in the1.1E2 and 1.1+2.1E2 groups increased,but these animals could provide the100%protection against virulent CSFV strains.Blood samples were collected at different time points after challenge,and copy numbers of CSFV were detected.Therefore,the 1.1E2and 1.1+2.1E2 subunit vaccines expressed from the recombinant baculovirus constructed in our laboratory could be used as candidate strains for the prevention of CSF.2.Construction and immunogenicity study of chimeric PCV2 VLPs vaccines with CSFV epitopeVLPs of chimeric PCV2 were generated by replacing the NLS of Cap with CSFV T-cell epitope（1446–1460 aa）,CSFV B-cell epitope（693–716 aa）,and CSFV T-cell epitope conjugated with B-cell epitope.Based on the crystal structure of Cap,one loop region in the middle of Cap was replaced with one B-cell epitope of CSFV.Subsequently,the bivalent vaccine was constructed,thereby providing a novel vaccine for the clinical prevention of the co-infection of CSF and PCV2.Based on the codon bias of the baculovirus,codon optimization and synthesis of genes of Cap-T,Cap-B,Cap-TB,and Cap-1T2B were performed.The synthesis of the modified target genes was conducted by Shanghai SANGON Biological Co.,Ltd.PCR reactions were performed using the synthesized gene fragments as templates,and amplified gene fragments were successively inserted into the transfer vector p FBD.Given the presence of three gene expression cassettes,each of the target genes was inserted into the transfer vector by three different pairs of restriction sites.Thus,four different recombinant transfer vectors were obtained,namely,p FBD-3-Cap-T,p FBD-3-Cap-B,p FBD-3-Cap-TB,and p FBD-3-Cap-1T2B.Four recombinant baculoviruses（Ac-3-Cap-T,Ac-3-Cap-B,Ac-3-Cap-TB,and Ac-3-Cap-1T2B）were obtained according to the operation manual of the baculovirus expression system.By indirect immunofluorescence and Western blot experiments,the results demonstrated that all the recombinant baculovirus constructs were efficiently expressed in the insect cells.Moreover,the cellular localization of these recombinant proteins was changed,as noted in both the nucleus and cell membrane.The target proteins expressed in insect cells,including Cap-T,Cap-B,Cap-TB,and Cap-1T2B,were purified by ultracentrifugation,and negatively stained with phosphotungstic acid.Visualization under the electron microscope revealed that Cap-T,Cap-B,and Cap-TB could form VLPs similar to PCV2,whereas Cap-1T2B could not be assembled into VLPs.To further explore the immunogenicity of the target proteins,the chimeric VLPs subunit vaccines were evaluated in mice and rabbits.The results indicated that all four chimeric VLPs vaccines induced the production of anti-PCV2 antibody and neutralizing antibody,but the level of neutralizing antibody in the Cap-1T2B group was significantly lower than that in the other groups.By contrast,the differences among the Cap-T,Cap-B,and Cap-TB groups（P>0.05）were not significant.Ig G for T-cell epitope was also induced in the Cap-T,Cap-TB,and Cap-1T2B groups,whereas Ig G for B-cell epitope was induced in the Cap-B,Cap-TB,and Cap-1T2B groups.Cellular immunity experiments confirmed that all the four vaccines could induce the cell proliferation response of lymphocytes to a certain degree.The serum level of IFN-γin mice in the Cap-B group was the highest,followed by that in the Cap-T and Cap-TB groups,the mean IFN-γlevels were 202.57,227.33 and 192.79 pg/m L,with no significant difference among the three tested groups（P>0.05）.The level of IFN-γin the Cap-1T2B group was quite low,which was significantly different from that in other groups（P<0.05）.Both groups of Cap-T and Cap-TB could induce specific CTL cytotoxic activity for T-cell epitope of CSFV and the mean CTL activity in the mice immunized with Cap-T and Cap-TB was 46.46%and 38.92%,respectively,at an E:T ratio of 50:1.in which the highest cytotoxicity was found in the Cap-T group.The experiments conducted in rabbits showed that the body temperature of all animals in all the immunized groups and control groups increased,and the regular type of CSF occurred when the duration was more than18 h.In addition,the existence of CSFV RNA was detected in the spleens in all rabbits in all the groups immunized with chimeric VLP vaccines.All the results indicated that chimeric VLP vaccines demonstrated a good response against PCV2,but the immunological response was insufficient to protect animals against CSFV.3.Construction and anti-FMDV study of transgenic pigs with overexpressed sIFITM3Plasmid psIFITM3 was linearized with the Acc I enzyme.Porcine fetal fibroblasts from large white piglets were transfected with a 1176 bp Acc I fragment containing sIFITM3 sequence from the psIFITM3 expression vector.Cell lines expressing sIFITM3were established.Transgenic pigs with sIFITM3 protein overexpression were obtained by manual cloning method.Wide distribution of the sIFITM3 gene in various tissues in transgenic pigs was detected by semi-quantitative measurement.The m RNA levels of the sIFITM3 gene in all the tissues（except for the liver）in transgenic pigs were significantly higher than those in the normal negative control pigs.The difference in tissues was roughly between 1.5-and fourfold,except for heart tissue,which exhibited the most significant difference of nearly 80-fold.Furthermore,the expression levels of sIFITM3protein in all the tissues in transgenic and negative control pigs were consistent with the corresponding m RNA levels.After being challenged with a virulent FMDV strain（O/CHA/2009）,the high body temperature and clinical symptoms of transgenic pigs manifested earlier than those of normal negative controls.Moreover,serious clinical signs were observed in transgenic pigs than in normal negative controls.The measurement of antibody and neutralizing antibody after challenge suggested that the levels of anti-FMDV antibody between transgenic pigs and normal negative controls were not significantly different.Measurement of copy numbers of FMDV showed that FMDV peaked 3 d after challenge and then became consistent thereafter,until it was completely undetectable at day 9 after challenge.In the negative control pigs,the highest FMDV number was found 5 d after challenge,followed with a gradual decline,but the copy number of FMDV was still detected in one of the pigs until 11 d after challenge.These results suggested no significant difference between the constructed sIFITM3-overexpressed transgenic pigs and normal negative controls of pigs against FMDV（P>0.05）. |
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