| Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that regulates proliferation and differentiation of hematopoietic progenitors. It has a potent effect on differentiation and maturation of dendritic cell (DC)as well as the expression of MHC and co-stimulatory molecules . Co-injection with plasmid DNA expressing GM-CSF has been reported to increase protective immunity induced by plasmid DNA.VP22, a tegument protein that has demonstrated the property of intercellular transport, is capable of distributing fused protein to many surrounding cells. It has been confirmed that it has adjuvant effect on vaccine.In this study, chicken GM-CSF and herpervirus VP22 were cloned and expressed and further used to explore their adjuvant efects on on DNA vaccine (IBV DNA vaccine).The main content is as follows:1.In this study, according to the gene sequence published in the GenBank (GenBank No: AJ621740), one pair of specific primer was designed. The Sichuan silky chicken GM-CSF cDNA was isolated from a concanavalin A (ConA)-stimulated peripheral blood lymphocytes using the reverse transcriptase-polymerase chain reaction (RT-PCR).The GM-CSF cDNA was ligated into the T vector and send to Sequence. Sequence analysis showed that the full-length cDNA is compose of 435 bp ,encoding an open reading frame of 144 amino acids with a predicted molecular mass of 16.15 kD . The deduced amino acid sequence of silky c hicken GM-CSF shares higher 98% and 15%-30% similarity to known GM-CSF in chicken species and other mammalian, respectively. The protein structure prediction showed that the GM-CSF protein has four a- helix and twoβ-strand and much coil. The percentage of a-helix andβ-strand and coil is 55.2%, 8.3%,36.5%,respectively. The c hGM-CSF structure is similar to that of mammalian GM-CSF.2.The GM-CSF without the signal peptide was amplified by PCR and ligated into prokaryotic expression plasmid pET-32a(+) to construct plasmid p32GM-CSF.After identification with restriction enzyme digestion and DNA sequencing,the p32GM-CSF was transformed into BL21 E.coil.The recombinant chGM-CSF was produced with induction by IPTG at 37℃.SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 34 kD in size. The acquired recombinant protein was purified by Nickel ion affinity chromatography and used to immunize rabbbit to prepare the Polyclonal antibodies (pAbs) specific for chicken GM-CSF .The ELISA assays showed that the antibody titre is very high and reach 1:3200. Western blotting analysis showed that it has good reactionogenicity and could bind with recombinant chGM-CSF protein specially expressed in E.coil.The acquired chGM-CSF protein and specific Polyclonal antibodies would offer critical material to the furt her research on biological activity of chGM-CSF.3.The full-length chGM-CSF,which was obtained from the previous constructed plasmid pGM-CSF, was ligated into the plasmid pVAX1.After identification by restriction enzyme digestion and DNA sequencing,the plasmid pVAX1-chGM-CSF was transfected into COS-7 cell by lipofectamine. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The recombinant chGM-CSF protein was prepared from COS-7 cell expressing supernatant 70 h after transfecting.The biological activity of chGM-CSF was measured by bone marrow cell proliferation assay. The results of restriction enzyme digestion and DNA sequencing showed that the plasmid pVAX1-chGM-CSF were constructed successfully. The mRNA transcription of chGM-CSF gene could be detected by RT-PCR in the COS-7 cells which were transfected with plasmids pVAX1-chGM-CSF. The expression of chGM-CSF protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids.The bone marrow cell proliferation assay showed that chGM-CSF protein could enhanced bone marrow cell proliferation obviously. This study would provide materials for the further development of DNA vaccine against IBV.4.According to VP22 gene sequence(GenBank No:L10283 ) already published in t he GenBank, one pair of specific primer was designed.T he VP22 gene was amplified from the MDV genome by PCR.Then, According to the sequence of IBV S1,M,N gene, the specific primer of S1,M,N gene were designed,respectively.The S1,M,N gene were amplified by PCR and ligated into the plasmid pVAX1 to construct recombinant plasmid ,respectively.After identification by restriction enzyme digestion and DNA sequencing,these recombinant plasmid were digested by two restriction enzyme,respectively and the big fragment of each recombinant plasmid was purified by agar electrophoresis. To construct fusion plasmid pVAX-VP2/S1 ,pVAX-VP22/M,pVAX -VP22/N,the each big fragment ligated with VP22 fragment dealed with same restriction enzyme,respectively.After identification by restriction enzyme digestion and DNA sequencing,the fusion plasmid pVAX-VP22/S1. pVAX-VP22/M, pVAX-VP22/N was transfected into COS-7 cell by lipofectamine,respectively. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The results of restriction enzyme digestion and DNA sequencing showed that these fusion plasmid were constructed successfully. The mRNA transcription of S1,M,N gene could be detected respectively by RT-PCR in t he COS-7 cells which were transfected with these fusion plasmids. The expression of S1,M,N protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with these fusion plasmids.This study would provide materials and foundation for the further evaluating the adjuvant effection of VP22 for IBV.DNA vaccine.5.In order to evaluate the immune-modulatory of VP22 and GM-CSF as a genetic adjuvant, the plasmids used for vaccination were extracted from E.coil and purified by a C hinese invention patent method which was established previously in our lab. The diluted plasmids were encapsulated by liposome in equal volume, and administered to the 7-day-old chickens by intramuscularly injection. After two weeks later, the chickens were boosted by DNA vaccine. Indirect ELISA methods and the Fluorescence activated cell sorter were used to detect anti-IBV antibody and the number of CD3+, CD4+and CD8+ T lymp hoctyes on day 0, 7,14,21,28 after immunization. Ten days after boosting, MTT assay were used to detect the proliferation level of immunized chicken.Two weeks after boosting, all chicken were challenged by virulent IBV strain and observed for two week. The results of specific antibody show that there was a significant difference (P＜0.05) in ELISA antibody levels between VP22 fusion group pVAX-VP22/S1 ,pVAX-VP22/M, pVAX-VP22/N and control group p VAX-S1,pVAX-N,pVAX-M since the 14t h-day after first inoculation. There was also a significant difference (P＜0.05 ) in ELISA antibody levels between chGM-CSF as molecular adjuvant group pVAX-chGM-CSF+pVAX-S1,pVAX-chGM-CSF+pVAX-N, pVAX-chGM-CSF+pVAX -M and control group since the 7th-day after first inoculation.The antibody level of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1.The results of lymphocyte proliferation showed that there was a significant difference (P＜0.05) between VP22 fusion group and control group.There was also a significant difference (P＜0.05)between chGM-CSF as molecular adjuvant group and contol group. The SI index of lymphocyte proliferation of pVAX-chGM-CSF+pVAX-S1 is 2.83,significant higher than those of pVAX-chIL-2+pVAX-S1,pVAX-chIL-18 +pVAX-S1.The percentage of CD4+, CD8+ T-lymphocytes from chickens immunized with VP22 fusion group was significantly higher (P＜0.05) than t hat of control group since the 21d after the first vaccination. W hen compared to that of control group,the significantly higher percentage of CD4+, CD8+ T-lymphocytes was observed in group immunized with chGM-CSF as molecular adjuvant since the 7th day after the first vaccination(P＜0.05).The percentage of CD4+, CD8+ T-lymphocytes of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+p VAX-S1, pVAX-chIL8+p VAX-S1. The result of virus-challenge assay showed that the protection rate of chGM-CSF as molecular adjuvant group was from 80% to 90%, higher than 75～80% of VP22 fusion group and 65～75% of control group.In all group, protection rate of the group immunized with pVAX-chGM-CSF+pVAX-S1 is highest and reach 90%,higher than 85%, 75% of pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1, respectively, far from 80% induced by inactivited vaccine. These results suggested that VP22 and chGM-CSF as a genetic adjuvant is feasible to enhance the immune response of IBV DNA vaccine .The chGM-CSF as molecular adjuvant is better than IL-2,IL-18 in the immune-modulatory of IBV DNA vaccine .These results provide a new way to develop effective avain vaccine.
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