| Objective Was analysised the molecular characteristics of porcine circovirus type 3 Liaoning isolate(LN-PCV3)and based on the significant economic loss of pig industry caused by the widely prevalent porcine(PCV2),especially,the status of greater economic loss by mixed infection,in this study,a univalent vaccine expressing PCV3 Cap gene and a bivalent vaccine expressing PCV2 and PCV3 Cap protein genes were constructed using baculovirus expression system,and the assessment of their immunogenicity was carried out to reserve a material basis for the integrated control of PCV2 and PCV3.Methods The specific primers were designed by reference to the Hubei isolate HB-PCV3-2016(Gen Bank accession number: KY954038.1).PCV3-positive samples by PCR were used as templates to PCR-amplify the whole gene,and after the PCR products were recovered using agarose gel with the kit,it was ligated with p MD-18-T vector,transformed into DH5 a competent cells,and the positive clones were screened for plasmid extraction,and the plasmid were identified by enzyme digestion.The correct plasmid was identified by enzyme digestion and sent to Shenggong biotech company to sequence.The sequenced gene sequences were analyzed by Mega7.0 and DNAStar software,and compared with the genetic sequences of other PCV3 isolates published on Gen Bank.On this basis,the Cap protein gene of PCV2 vaccine strain PCV2-SH(accession number: HM038027.1)and the Cap protein gene of LN-PCV3 were selected,respectively and the PCV2 Cap protein gene was linked and fused to the PCV3 Cap protein gene by the rigid linker peptide,as an immunogenic gene.According to the codon preference of insect cells,the immunogenic gene was artificially modified,optimized and synthesized,the immunogenic gene PCV2-3-Cap was obtained and cloned into p MD-18-T vector to construct recombinant plasmid T-PCV2-3-Cap.Specific primers were designed with the optimized PCV3-Cap protein gene and PCV2-3-Cap fusion protein gene by,and PCV3-Cap and PCV2-3-Cap protein genes were amplified by T-PCV2-3-Cap as template.Then,it was transformed into DH5 a competent cells,and the positive clones which were screened were selected for plasmid extraction and identified by enzyme digestion.The PCV3-Cap gene and the PCV2-3-Cap fusion gene with the correct identification result were cloned into the p Fast Bac 1 vector,and the baculovirus transfer vectors p Fast Bac-PCV3-Cap and p Fast Bac-PCV2-3-Cap were separately constructed.p Fast Bac-PCV3-Cap and p Fast Bac-PCV2-3-Cap were transformed into DH10 Bac competent cells,respectively,.After screening for blue and white spots,recombinant bacmids were extracted and transfected into SF9 insect cells,PCV3 Cap was obtained by screening gene,which was recombined with the recombinant baculovirus of PCV2 and PCV3 Cap,After PCR,SDS-PAGE,Western blot,immunofluorescence,electron microscopy and other experimental methods,the virus was purified by sucrose density centrifugation.The purified virus was used as an immunogen.BALB/c mice were immunized with 1×108 pfu of recombinant baculovirus rv Ac-PCV3-Cap and rv Ac-PCV2-3-Cap,and the control group was set up,immunized every 2 weeks,and immunized three times.0d,14 d,28d,42 d after the first immunization,collect blood from the tail of the mouse respectively and serum was separated.The antibody levels of PCV2 and PCV3 were detected by ELISA.42 d after the first immunization,the mice were sacrificed and the spleen was removed and calculated the lymphocyte proliferation index SI of mice in each immunization group by CCK-8.According to the method of the cytokine detection kit,IL-2,IL-4,IFN-γ and other cytokines of the immunized mice were detected to verify the expression of the PCV3-Cap protein gene and the PCV2-3-Cap fusion protein gene in the baculovirus system and its immunogenicity.Results The PCV3 full-length sequence was cloned from PCV3 positive sample by PCR,which was 2000 bp in length and named LN-PCV3(Gen Bank accession number: MH177453.1).Genetic phylogenetic tree analysis showed that the current PCV3 isolates in China can form three branches(3a cluster,3b cluster and 3c cluster),and LN-PCV3 strain was in the 3a cluster branch,which has the highest homology with the Hubei strain HB-PCV3-2016(Gen Bank accession number: KY354039.1),reaching 99.7%.On this basis,the recombinant baculovirus transfer vectors p Fast Bac-PCV3-Cap and p Fast Bac-PCV2-3-Cap were transformed into DH10 Bac competent cells for transposition,respectively.Was confirmed by PCR cross check the obtained recombinant granules r Ac Bacmid-PCV3-Cap and r Ac Bacmid-PCV2-3-Cap,then transfect the recombinant bacmid into SF9 insect cells to prepare recombinant baculovirus.The recombinant baculovirus was serially passaged 3 times,and when the recombinant baculovirus rv Ac-PCV3-Cap and rv Ac-PCV2-3-Cap were passaged 3 times(P3 generation),the pseudoviral particles and baculovirus particles were observed by transmission electron microscopy.The M13 universal primers,PCV3-Cap gene-specific primers and PCV2-3-Cap gene-specific primers were used to amplify by PCR.The target fragments were observed at 674 bp and 2974 bp,1514 bp and 3814 bp,and the sizes were consistent with the theoretical values.The target bands appeared at 30 k D and 55 k D by SDS-PAGE and Western blot.The immunofluorescence experiments showed that the two groups of recombinant proteins showed green fluorescence under fluorescence microscope,The normal cells showed no fluorescence effect,which proved PCV3-Cap gene and PCV2-3.Cap fusion gene were expressed in SF9 cells and the recombinant baculovirus rv Ac-PCV3-Cap and rv Ac-PCV2-3-Cap were successfully screened.The results of antibody level test showed that the levels of PCV3 antibody in rv Ac-PCV3-CAP and rv Ac-PCV2-3-Cap immunized groups were significantly higher than PBS negative control group and PCV2 inactivated vaccine positive control group(P < 0.05),but here was no significant difference in PCV3 antibody levels between the rv Ac-PCV3-CAP and rv Ac-PCV2-3-Cap immunized groups(P > 0.05),the rv Ac-PCV2-3-Cap immunization group had significantly higher PCV2 antibody levels than the PBS negative control group and rv Ac-PCV3-Cap group and the difference was significant(P < 0.05),but there was no significant difference in antibody levels when compared with the PCV2 inactivated vaccine-positive control group(P > 0.05).The results of CCK-8 assay for spleen lymphocyte proliferation index(SI)of the mice showed that rv Ac-PCV3-Cap,rv Ac-PCV2-3-Cap immunization group and PCV2 inactivated vaccine positive control group were compared with PBS group,SI Index was significantly different(P < 0.05),but the difference was not significant between the other three immunized groups(P > 0.05).The detection of cytokine levels showed that the injection of recombinant baculovirus group could stimulate the body to increase the secretion levels of IL-2,IL-4 and IFN-γ,which was not significantly different from the PCV2 inactivated vaccine group(P > 0.05),but was significantly higher in the PBS negative control group(P < 0.05).Conclusions 1.LN-PCV3 complete genome was successfully cloned and its molecular biological characteristics were analyzed.2.The expression of PCV3 Cap protein gene and PCV2,PCV3 Cap protein fusion gene was achieved by baculovirus expression system,and recombinant baculovirus rv Ac-PCV3-Cap and rv Ac-PCV2-3-Cap were successfully screened.3.Mouse immunization experiments showed that recombinant baculovirus rv Ac-PCV3-Cap and rv Ac-PCV2-3-Cap can stimulate the body to produce specific humoral and cellular immune responses,providing technical support and material reserves for the development of novel porcine circovirus vaccines. |
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