| The core pathogenicity-related-1(PR1)domain of activation-related secretary protein(Ov-ASP-1)of Onchocerca volvulus has potent adjuvant effect.However,previously reported recombinant PR1 was expressed as His-tagged protein which requires expensive nickel column for purification with shorter half-life due to its small molecular mass.In this study,Ov-ASP-1 PR1 was expressed as elastin-like polypeptide(ELP),ELK16 self-aggregating peptide or His tag fusion protein and used to immunize mice using recombinant VP2 protein of infectious bursal disease virus(IBDV)as the model antigen,aiming at simplification of recombinant PR1 purification and clarification of influence of fusion tags on PR1 adjuvant activity.First,the coding sequence for IBDV VP2 segment was adapted to E.coli codon usage with tobacco etch virus(TEV)protease recognition site introduced at the 5' end and the synthetic sequence was cloned into ELP fusion expression vector.The recombinant pELP-VP2 was transformed into E.coli and the expression of fusion protein was induced with IPTG The results showed that the expressed fusion protein had an expected molecular weight of 67 kDa.Optimization study showed that phase transition temperature of ELP-VP2 fusion protein was 28 C in the presence of 2 M NaCl.The ELP-VP2 fusion protein was purified to 96%purity with a yield of 24 mg/L after one cycle of inverse transition cycling(ITC).By incubation with the active inclusion body of TEV protease,ELP tag was efficiently cleaved with 86%cleavage efficiency.After removing ELP tag by additional cycle of ITC,the recovered VP2 protein had 95%purity and 24mg/L yield which could be recognized by VP2-specific antibody.These data suggest that ELP is an efficient purification tag of recombinant proteins and the recombinant VP2 protein can be further developed as novel IBD subunit vaccine.Then,the coding sequence for ASP-1 PR1 domain was inserted into His-tag fusion expression vector pET-30a,ELP fusion expression vector pET-ELP or ELK 16 fusion expression vector pET-P16P.The recombinant vectors were transformed into E.coli and expression of the three fusion proteins was induced with IPTG The results showed that the expressed ELP-PR1,ELK16-PR1 and His-PRl fusion proteins had expected molecular weights of 60,28 or 23kDa,which were purified to 92%,95%or 97%purities by ITC,centrifugation and nickel affinity chromatography,with production yields of 38,96 and 134 mg/L,respectively.Finally,mice were immunized with VP2,VP2+IFA,VP2+ELP-PR1,VP2+ELK16-PR1 or VP2+His-PR1 and boosted on day 14 post immunization(dpi).The serum samples were collected on 7,14,21 and 28 dpi and the VP2-specific IgG,IgG1 and IgG2c were determined by indirect ELISA.The splenic lymphocytes were harvested on 28 dpi for splenic lymphocyte stimulating index and cytokine detection.Compared to that in VP2 immune serum,the VP2-specific IgG level was significantly higher in His-PR1(p<0.05),ELP-PR1(P<0.01),ELK16-PR1(P<0.01)or IFA(P<0.01)adjuvant group,with the highest IgG level in ELP-PR1 adjuvant group on 7 dpi.The specific IgG1 was significantly higher in ELP-PR1(p<0.05)and ELK16-PR1(p<0.01)adjuvant groups,but not in His-PR1 and IFA adjuvant groups,with the highest IgG1 level in ELK16-PR1 adjuvant group.The specific IgG2c levels in all of four adjuvant groups were significantly(p<0.01)higher than that in VP2 immunization group with the highest IgG2c level in ELP-PR1 adjuvant group.On 28 dpi,the specific IgG levels of all four adjuvant groups were significantly(p<0.01)higher than that in VP2 immune serum,with the highest IgG level in ELP-PR1 adjuvant group.The IgG1 level was significantly higher in His-PRl(p<0.05)and other three adjuvant groups(p<0.01),with the highest IgG1 level in ELP-PR1 adjuvant group.The specific IgG2c levels in the four adjuvant groups were significantly(P<0.01)higher than that in VP2 immune serum,with the highest levelin ELP-PR1 adjuvant group.The averaged splenic stimulation indexes of VP2,VP2+IFA,VP2+ELP-PR1,VP2+ELK16-PR1 and VP2+His-PR1 groups were 9.11±0.32,11.04±0.57,28.9±0.3,12.06±0.24 and 10.86±0.11,respectively.Compared to VP2 immunization,splenic stimulation index was significantly(P<0.01)higher in ELP-PR1 adjuvant group,but not in other three immunization groups.Compared to VP2 immunization,the IFN-y level was significantly(P<0.01)higher in ELP-PR1 adjuvant group,but not in other three immunization groups.The IL-6 level was significantly higher in ELK16-PR1(P<0.05)and ELP-PR1(P<0.01)adjuvant groups,but not in His-PR1 and IFA adjuvant groups.The IL-10 levels were also significantly higher in ELP-PR1(P<0.01)and other three immunization groups(P<0.05).TNF-a levels were significantly(P<0.01)higher in three PR1 adjuvant groups,but not in IFA adjuvant group.These data suggest that ASP-1 PR1 protein with different fusion tags remain adjuvant effects. |
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