Immune Adjuvant Effect Of Mycobacterium Tuberculosis Hsp70 On PCV2 Cap Protein

Porcine circovirus type 2(PCV2)is the main causative agent of porcine dermatitis and nephrotic syndrome(PDNS)and post-weaning multisystemic wasting syndrome(PMWS).The prevalence of PCV2 has caused huge economic losses to the swine production worldwide.Presently,the prevention and control of PCV2 relies mainly on vaccination.The commonly sued vaccines include whole virus inactivated vaccine,recombinant chimeric vaccine and subunit vaccine.However,the virus replicates poorly in PK-15 cells,and thus the cost for inactivated vaccine preparation is relatively high.The virus-like particle vaccine can be expressed in E.coli and insect cells,which requires complex procedure for purification.Therefore,it is ungent to develope inexpensive novel PCV2 subunit vaccine.The self-aggregating peptide ELK 16 can form active inclusion bodies in E.coli and the fusion protein can be purified by centrifugation.Mycobacterium tuberculosis Hsp70 is an important pathogen-associated molecular pattern with an immunoadjuvant effect due to stimulation of innate immune cells to release cytokines such as TNFa,IL1,IL6,IL10,and IL-12.To simplify the preparation process of PCV2 subunit vaccine and Hsp70 molecular adjuvant,in this study ELK16 was used as a fusion tag for the expression and purification of PCV2 Cap protein and Mycobacterium tuberculosis Hsp70.By using mice as an animal model,the adjuvant effect of Hsp70 for recombinant Cap protein was investigated.The ELK 16 coding sequence was fused to Mycobacterium tuberculosis Hsp70 and PCV2 Cap genes,and the recombinant vectors were transformed into E.coli,respectively.After induction with IPTG,the fusion proteins ELK16-Cap,ELK16-Hsp70 and ELK16-Cap-Hsp70 were purified by centrifugation and repeated washing under optimized conditions.The fusion proteins purified had the purity of 95%,96%and 85%,with the yield of 92 mg/L,84 mg/L,and 83 mg/L bacterial culture,respectively.Thirty mice were divided to five groups,and immunized with ELK16-Cap,ELK16-Cap + incomplete Freund's adjuvant(IFA),ELK16-Cap +ELK16-Hsp70,ELK16-Cap-Hsp70 or PBS as the control.At day 7,14,21 and 28 post primary immunization,serum samples were collected and Cap-specific antibodies were detected by indirect ELISA.The results showed that,on day 7 after primary immunization,only IFA +ELK16-Cap and ELK16-Hsp70 + ELK16-Cap immunization groups were positive for Cap antibodies.From day 14,the specific antibody levels of the four immunized groups increased gradually,among which ELK16-Hsp70 + ELK16-Cap immunized group had the highest antibody level,followed by IFA + ELK 16-Cap immunized group and ELK16-Cap immunization group.From day 21,the specific antibody levels of the four immunization groups further increased,among which ELK16-Cap-Hsp70 immunized group had the highest antibody level.Two weeks after boosting immunization,spleen lymphocytes were collected from immunized mice and cultured in 96-well plates.After stimulation ConA or ELK16-Cap,the cytokine expression was detected.The results showed that TNF-? concentrations of of PBS control group,ELK16-Cap,ELK16-Cap + IFA,ELK16-Hsp70 + ELK16-Cap and ELK16-Cap-Hsp70 immunization groups were 588.55,802.55,995.55,1382.55 and 1719.55 pg/mL,respectively.The IFN-y concentrations of the five groups were 46.30,92.22,155.56,470.37 and 518.15 pg/mL,respectively.IL-12 concentrations of the five groups were 14.72,28.06,20.83,31.39 and 34.72 pg/mL,respectively.These data indicate that the Hsp70 has a better stimulatory effect on PCV2 Cap ELISA antibody production than IFA.The Hsp70 fused to Cap protein had stronger stimulation effect on TNF-?,IFN-? and IL-12 production than ELK16-Hsp70 + ELK16-Cap.To optimize the antigenicity of PCV2 recombinant antigen,the His tag was fused to the neutralization epitope of PCV-2a,PCV-2d,PCV-2e and PCV-2b Cap gene(4Cap)and/or Hsp70 gene.The fusion proteins 4Cap-His,Hsp70-His and 4Cap-Hsp70-His were purified using affinity chromatography.Thirty mice were divided into five groups:PBS control group,4Cap-His immunization group,4Cap-His+ Hsp70-His immunization group,4Cap-Hsp70-His group,and 4Cap-His + IFA immunization group.Serum samples were collected in 7-day intervals after primary immunization,and Cap-specific antibodies were determined by indirect ELISA.Splenic lymphocytes were collected from immunized mice at day 14 after boosting immunization,and cytokine concentrations were measured using commercial kits.On day 14 post boosting immunization,three mice from each group were challenged with 2x103TCID50 PCV2b,and serm samples were collected for viremia detection on day 3 or 7 post challenge.The results showed that on day 7 after primary immunization,only 4Cap-His + Hsp70-His and 4Cap-Hsp70-His immuniztion groups were positive for ELISA antibody.By day 14,the control and 4Cap-His immunization groups were still negative for anti-Cap antibodies,4Cap-His + IFA immunization group was positive for anti-Cap antibody but lower than that of 4Cap-Hsp70-His immunization group.From day 21,the antibody levels of four immunization groups increased significantly,among which antibody level of 4Cap-Hsp70-His immunization group was the highest.Compared to the control group,lymphocytes expression levels of TNF-a,IFN-y,and IL-12 in the four immunization groups were significantly higher than those of concanavalin A stimulation group.The viremia of all four immunization groups were significantly decreased on day 7 post challenge,with the highest decrease in 4Cap-Hsp70-His immunization group,followed by 4Cap-His plus Hsp70-His,4Cap-His plus IFA and 4Cap-His immunization group.These results indicate that the ELK16 fusion tag has no significant effect on Hsp70 stimulation of Cap-specific ELISA antibody production.Fusion proteins ELK 16-Cap-Hsp70 and 4Cap-Hsp70-His can stimulate better humoral immune response and cytokine production than IFA,Hsp70 fusion PCV2 Cap protein can stimulate better than Hsp70 plus PCV2 Cap.