| In this study, the small circovirus-like particles were observed by transmission electron microscope through negative straining in the homogenate of lung,liver,spleen,intestines and lymph nodes,origined from the pig skeptically affected by postweaning multisystemic wasting syndrome in some farms of Inner-mongoliar. PK-15 and Vero cells cultures were simultaneously inoculated with the above mixed homogenate;after 3 or 4 days,specific PCV2 DNA sequences were detected in both cultures by PCR; Moreover, PCV2-related genes can be observed from the first generation PK-15 culture,which indicates that the PCV2 strain is more sensitive to PK-15 than to Vero.After PK-15 cultures contained PCV2 were continuously cultured seven generations, ±17nm-diameter icosahedral virions can be observed under transmission electronic telescope either by negative staining or by ultrathin section. After configurating PK-15 cell cultures contained PCV2 adopted isodensity gradients in CsCl solution, The virions of porcine circovirus 2 can,t be observed under electron microscopy ;However,about 36kDa and 26kDa protein blots can be found through Western blot. PCR was used to amplify the genetic fragments of PCV2 from the tissues homogenate.The whole length is 1767bp.The full genome of Inner-monogolia strain(NM2002)of PCV2 has been cloned.This is the first confirmation that the PCV2 have been isolated from swine loads in Inner-monongolia,and even the sequences of the full genome between NM2002 and the published one(AF055394)shows 99.8% homology.The interested genes ORF2 and ORF1 were respectively gained from the formed genomic library and swine IL18 gene was gained from recombinant plamid stored in our laboratory. The eukaryotic expression vector pVAX1 was used to construct the nucleic acid vaccine plasmids,They are pVAX1-ORF2,pVAX1-ORF2ORF1,pVAX1-IL18ORF2 and pVAX1-IL18ORF2ORF1 contained capsid,replicon gene of PCV2 and the swine cytokine gene of IL-18, respectively. The recombinant plasmids were transfected into BHK21 cells via liposome, and the transcription and expression of interest genes were detected by RT-PCR, indirect immunofluorescent assay and Western blot. The results showed that the transcription of interest genes could be detected from the cells transfected by the recombinant plasmids ,and these recombinant plasmids could express the interest proteins in vitro. BALB/c mice were immunized by the four recombinant plasmids separately .The detected immunological index manifested that the spleen T lymphocyte subgroups(CD4+,CD8+) examined in four experimental groups have more significant total numbers than the control groups,but no differences between the experimental groups.Meantime the specific antibody againstPCV2 could be induced by nucleic acid vaccine plasmids;However, there is no sense in statistics,compared with the control groups.In addition, cellular and humoral immunity caused by nucleic acid either with IL18 gene or not, don't bring any differences,may be which is the result from different species.Relatively ,the nucleic acid named by pVAX1-IL18-ORF2-ORF1 is suprior. Two recombinant expression plasmids of pUTA-P1-ORF2 and pUTA-P1-ORF2ORF1 were constructed by inserting capsid gene alone and both capsid and replicon fusion genes of PCV2, and the precursor structural protein P1-2A gene of O type FMDV downstream of combined promotor (ATI-P7.5) and tandomly repeated promotor (16×P7.5) of FPV expression vector pUTAL respectively. The recombinant plasmids and FPV 282E4 wild strain were co-transfected into CEF cells and the homologous recombination occurred mutually. Thus,two recombinant viruses vUTA-P1-ORF2 and vUTA-P1-ORF2ORF1 were selected and confirmed by RT-PCR, , indirect immunofluorescent assay and Western blot. BALB/c mice were immunized with the recombinant viruses by muscular inoculation, and the level of serum antibodies and the numbers of spleen T lymphocyte subgroups were examined after three times inoculation. The results showed that both of the two recombinant fowlpox viruses could increase the percentage of the T cell subgroups which were significantly higher than that of the control virus。The immunized mice could produce the sera antibodies against both PCV2 and O type FMDV in some degree.After comparation,the recombinant virus pUTA-P1-ORF2ORF1 was determined as candidate. To approach the feasibility of different united ways of immunization, the recombinant fowlpox virus vpUTA-P1-ORF2ORF1 and the nucleic acidvaccine pVAX1-IL18-ORF2-ORF1 were used to immunize the swine in "prime-boost"programme.We observed that priming the swine with DNA plasmid pVAX1-IL18-ORF2-ORF1,followed by boosting with the recombinant virus vpUTA-P1-ORF2ORF1 produced strongly cellular immunity and humoral immunity.During experiment,the antibody level produced by PCV2 was lower than that of O type FMDV,1:40 and 1:400 respectively;but their antibody kinetics were compatible.The two experimental groups ,especially which first inoculating pVAX1-IL18-ORF2-ORF1 then vpUTA-P1-ORF2ORF1, had considerably higher CD4+/CD8+ T lymphocytes subgroups compared with the control groups.Whether the ratio between effective cells and target cells was 50:1 or 25:1,the specific CTL of experimental groups had much more significant differences with the control (FPV),even still the group of primine nucleic acid vaccine boosting recombinant virus had the most brave cytotoxicity of specific CTL;Moreover the E/T ratio of 50:1 is more excellent.In whole the experimental process,six lymphoproliferative reaction assays were carried and T lymphocytes cultures were stimulated with nonspecific ConA and specific protein and live virus of FMDV.The result indicates that the proliferative index of trial groups increased more strikingly than the control one.Besides,the specific stimulates with live virus led better prolifiration than which with protein,while nonspecific proliferative index was also slightly higher than that of the specific protein stimulate.Therefore,the conclusion is drawn that the prokaryotic expression protein ,though renatrued,is still inferior stimulating activity to live virus. In a word,the immunization programme here ,in which pVAX1-IL18-ORF2-ORF1 DNA vaccine inoculated firstly and... |
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