The Expression Of Porcine IL-10 And The Immunological Enhancement Of Exogenous Porcine IL-10 Active Immu-Nization On Inactivated PRRSV Vacine

Interleukin-10 (IL-10) is mainly produced by Th2 cells,which can suppress the Thl cells to produce immune regulatory cytokine.The porcine IL-10 (pIL-10) is closely related to the infection of porcine reproductive and respiratory s-yndrome virus (PRRSV) which seriously threatens the swine industry.Some res-earches showed that the infection of PRRSV could induce the increase of pIL-10 expression and the induction of the pIL-10 expression was one strategy of PRRSV to regulatory the immune system of the host.The purpose of the study is to investigate whether the IL-10 antibody induced by exogenous IL-10 vacci-ne could eliminate the immunosuppression of endogenous pIL-10 and enhance the immune function of host or vaccine immunogenicity.1. The construction,expression and identification of recombinant eukaryotic expression plasmid for pIL-10 full length cDNAFirstly,the peripheral blood mononuclear cells (PMBC) of porcine were se-parated and stimulated by ConA for 48h.The 528bp cDNA encoding open rea-ding frame of porcine IL-10 was then cloned by RT-PCR from PMBC with s-pecific primes based on the reported sequence in GeneBank (Accession number: L2001),inserted into pMD18-T plasmid,then identifieded by enzyme digestion,P-CR and sequence identification.The 528bp frame was connected to eukaryotic expression plasmid pVAX1 and identified by enzyme digestion,PCR and seque-nce identification. The recombinant plasmid pVAX1-pIL-10 was constructed and the recombinant bacterium was named with pVAXl-pIL-10.2. The construction,expression and identification of recombinant procaryotic expression vector for pIL-10The specific primers were designed to sub-clone the fragment encoding m-ature pIL-10 without signal peptide from the recombinant plasmid pVAXl-pIL-10.The fragment was inserted into pMD18-T plasmid after cloned by PCR.The recombinant plasmid pMD18-T-pmIL-10 was identified by enzyme digestion, P-CR and sequence identification.The 490bp frame was cloned from recombinent plasmid pMD18-T-pmIL-10,inserted into plasmid pET32-a(+)and identified by e-nzyme digestion,PCR.The recombinant plasmid pET32-pmIL-10 was translated i-nto E.coli Rosetta to express IL-10 protein for analysis.The result of SDS-PAG E showed that the recombinant protein pIL-10 was about 35KD and expressed mainly in sediment.The optimization of expression was carried out and the ex-pression was higher at 0.2mM IPTG in 4h.The recombinant protein pIL-10 was purified with His-tag purification kit.The antiserum was separated from the blo-od of pigs immunized with the purified recombinant protein pIL-10 emulsifiedb y oil emulsion adjuvant.The antibody titer was tested by ELISA,P/N=8.The res-ult of western-blot showed that there was a specific strap at the purpose strap. The result of the study showed that the immunity of recombinant protein pIL-10 and the reactivity of anti-pIL-10 antibody induced by recombinant protein were very good.The recombinant eukaryotic expression plasmid pVAX1-pIL-10 was transfe-cted into COS-7 cells with liposome 2000 for 24-48h.The transcription of pIL-10 mRNA was identified by RT-PCR and the expression of pIL-10 gene was identified by indirect immunofluorescence assay (IFA) with auto-made antibody induced by recombinant protein pIL-lO.The result showed that the recombinant eukaryotic expression plasmid pVAX1-pIL-10 could express the pIL-10, the au-to-made anti-pIL-10 antibody had good activity with the recombinant protein.T he recombinant protein pIL-10 could induce pigs to produce anti-pIL-10 antibo-dy.3. The establishment of TaqMan real time fluorescent quantitativity assay for pIL-10 mRNAA pair of primers and probe were designed based on the recombinant pl-asmid pVAX1-pIL-10.The conditions of reactivity were optimized.The TaqMan real time fluorescent quantitativity stand curve was established at the optimized condition and the methodology was evaluated.Its R2 was 0.99,its was efficiency was 2.0.The sensitivity of the assay was 10copies/ul and the specificity and re-petitiveness of the assay was very good.The gene of pGAPDH was cloned by RT-PCR and inserted into pMD18-T.The stand curve was established as the re-combinant plasmid pMD18-T-pGAPDH as stand template at the optimized co-ndition and the methodology was evaluated.Its R2 was 0.99, its efficiency was 2.0.The sensitivity of the assay was 1000copies/ul and the specificity and repe-titiveness of the assay was very good.4. The effection of anti-pIL-10 antibody to the pIL-10 mRNA expression of PMBCS in vitro testThe objective of this study in the fourth chapter was to value the effecti-on of anti-pIL-10 antibody to the pIL-10 mRNA expression of PMBCS in vitr-o test.The PMBCS were separated,diluted into 106/ml and spreaded into each hole in 500ul/hole.The anti-serum was diluted in 10,20,40,80 times and spr-eaded into the cell plate in 500ul/hole,each dilution has 4 replications.The neg-ative serum control and the cell control were set.When 2.5ul ConA (lmg/ml) was added into each hole, the cell plate was put into the cell culter incubator for 48h.Then the total RNA was extracted from the lymphocytes and coloned by RT.The expression of pIL-10 was tested with real-time PCR established in chapter 3.The result showed that the expression of pIL-10 mRNA was higher in 10 time dilution than other groups and there was no obvious difference bet-ween the 20,40,80 time dilution groups and the cell control group.The anti-pIL-10 could stimulate the lymphocytes to produce pIL-10 mRNA and the eff-ect was in dose dependent manner.5. The influence of pIL-10 to the immune efficiency of inactivated PRRSV vaccine4 weeks pigs were immunized intramuscularly with inactivated PRRSV va-ccine with recombinant plasmid pVAXl-pIL-10 encoding the pIL-10 protein or purified recombinant protein pIL-10.simultaneously, control group was setup.The serum was pooled from the pigs and the level of antibody titer was assessed by ELISA the levels of neutralizing antibodies was assessed by virus neutraliz-ed test (VNT).The subpopulations expressing CD4+、CD8+、CD3+ were detected by flow cytometry.It was showed that the high titer anti-pIL-10 antibody was produced in the group immunized with recombinant protein in advance of or s-imutinously with inactivated PRRSV vaccine,but not in other groups;the increa-sing anti-PRRSV antibody was produced only in the group immunized with rec ombinant protein in advance,but low titer were produced in other groups.The n-eutralization tests showed that the level of neutralization antibody titer of the test teams was very low;The flow cytometry showed that the CD4+/CD8+ of the test teams was lower than that of the control team,but there were no obvious differences bet-ween the test teams.