Expression And Some Immunological Characteristics Of Reticuloendotheliosis Virus Gp90 Protein

ObjectiveReticuendotheliosis virus(REV) is a member of gammaretrovirus with a varity of strains,which can causes oncogenic,immunosuppressive and runting syndrome in multiple avian hosts including chickens,ducks,turkeys and some other birds species.REV causes immunosuppression in infected chickens,resulting in an increased susceptibility to concurrent or secondary bacterical and viral infections and poor immune response to other vaccines.REV have a world-wide distribution,it reported in Britain, Germany, Australia,the trends in China have a tendency to expend,its rate of infection in poultry in China has reached 20% to 30%,caused severe damage to the poultry industry.REV is single-stranded RNA genome consisting of gag, pol, and env genes,flanked by long terminal repeats(LTRs).env gene encoding the gp90 protein and gp20 protein, gp90 protein belongs REV envelope protein, is REV immune immunogenic proteins, can induce the body to produce neutralizing antibodies, play an important role in the body’s immune response. Thus, obtained the gp90 protein in vitro,study its immunological characteristics, for study REV diagnosis and prevention,pathogenic mechanisms have great significance.MethodsThe test by selecting pET-22b(+) and pET-30a(+) carriers, designed primers under the whole gp90 gene and added EcoR I and XhoI restriction enzymes inprimers.Genomic DNA extracted from pMD-19T-gp90 recombinant bacteria was used as the PCR template.The gp90 gene was amplified by PCR and EcoR I and XhoI sites were introduced at the beginning and end of the PCR products by primers. Double digested PCR products were ligated into pET-22b(+) and pET-30a(+) which double digested by EcoR I and XhoI. The resulting constructs were transformed into E. coli DH5α,identified by PCR and double digestion positive recombinant plasmid sent to nucleotide sequencing. Use the online software of NCBI blast to analysis the sequencing results,sequencing results of comparative analysis, to observe whether the gene mutation.The pET-22b(+)-gp90 and pET-30a(+)-gp90 recombinant plasmid was transformed into E. coli BL21(DE3), identified by PCR and double digestion positive recombinant plasmid sent to nucleotide sequencing again.Use the online software of NCBI blast to analysis the sequencing results again,especially the gene mutation.then using IPTG to induce expression of protein,identified target protein expression and expression form,reactogenicity by SDS-PAGE and western-blot, the target protein was purified preparation chicken anti-gp90 serum, the immunized chickens serum were collected at 21,28,35,42 days, ELISA detected antibody titer. Chicken sacrificed at 42 days to prepare spleen lymphocytes, gp90 antigen detected after in vitro stimulation cytokine secretion(IFN-γ, IL-4), analysis of cellular immune responses.Results1. Successfully clone gp90 gene segment and construct recombinant plasmid pET-22b(+)-gp90 and pET-30a(+)-gp90, through IPTG induction, when 37 ℃, 1mM IPTG, pET-22b(+)-gp90 and pET-30a(+)-gp90 recombinant proteins express efficiently.The recombinant proteins corresponded to their predicted molecular masses by SDS-PAGE.,The expected size is 43 kDa.But when 30℃,recombinant proteins express poor.Western-blot analysis showed that the protein expressed in inclusion bodies.2.Purification experiment shows that the gp90 recombinant proteins can be purified well.The purpose of the purified protein purity above 95%;ELISA detected gp90 antiserum titer is 12500 EU. on 42 d gp90 antiserum immune antibody titer reached the highest.3. Cytokines detection results showed that IFN-γ and IL-4 concentration were114.56 pg/mL, 68.76 pg/mL, which significantly higher than PBS negative control group, the results showed that gp90 can stimulate the chicken to produce higher levels of IFN-γ and IL-4, concluded after gp90 protein immune chicken can stimulate Th1 andTh2 cells in the immune response.ConclusionStudy have Successful clone and expression of gp90, and confirm gp90 has good immunogenicity and reactionogenicity. Additionally, chicken can produce immune response of helper T cell 1 and 2(Thl and Th2).