Immunogenic Analysis Of Polymers Formed By Truncated Calreticulin Fused With FMDV VP1 And PCV2 Cap Protein

Foot-and-mouth disease?FMD?is a highly contagious disease of cloven-hoofed animals which pathogen is Foot-and-mouth disease virus?FMDV?,the structural protein VP1 is the most important antigenic protein.Porcine circovirus disease?PCVD?is an important immunosuppressive pathogen that endangers the large-scale pig industry in the world,which pathogen is Porcine circovirus type 2?PCV2?.Cap protein is the nucleocapsid protein of virus.Calreticulin?CRT?is a 46 kDa length Ca²⁺binding protein of endoplasmic reticulum cavity.Full-length or shortened CRT can effectively promote protein folding and polymerization.CRT fusion exogenous proteins can also maintain the polymers'structure and good immunogenicity.At present,vaccination is still the most effective way to prevent FMDV and PCV2 infection.In order to prepare a safe and effective subunit vaccine of FMDV and PCV2,VP1 protein of FMDV serotype O and Cap protein of PCV2 were respectively used to fuse with different lengths of truncated CRT for expression into polymers.The immune potential of the vaccine was tested in mice and guinea pigs.The main results are as follows:1.The full-length of VP1 protein was used to fuse different lengths of truncated CRT?120-250 aa/120-308 aa?at the N and C terminal respectively.Four different forms of VP1-CRT fusion proteins?rV4C,rC4V,rV5F and rF5V?were successfully constructed.In order to promote the correct folding of VP1-CRT fusion proteins,co-expression of chaperone protein Tf16 was adopted to achieve soluble expression.VP1-CRT fusion proteins were successfully purified by gel filtration method,and verified that all of them could form into polymers.Dynamic Light Scattering?DLS?and other methods showed that VP1-CRT fusion proteins could form large-particle proteins with a diameter of about 100 nm.Indirect ELISA method was used to analyze the antigenicity of VP1-CTR fusion proteins,the results showed that they could all specifically identify clinical positive serum of FMDV serotype O,anti-FMDV mAbs 2D2 and anti-pep?VP1 141-160 aa?mAbs 3D-A11 which indicated that they all had good reactivity.2.Purified VP1-CTR fusion proteins were combined with adjuvants to prepare vaccines.Immunization of mice could elicit high levels of antibodies,which could recognize naked VP1 protein and FMDV through indirect and sandwich ELISA,respectively.Guinea pigs were immunized with VP1-CRT fusion proteins,and the results of indirect ELISA and sandwich ELISA showed that they produced high levels of humoral immune,and there was no difference between rC4VZ group and inactivated vaccine.Meanwhile,the levels of IFN-?,IL-10,IL-18,TNF-?,GM-CSF and lymphocyte proliferation were detected.The adjuvant Montanide^TM ISA 50V2and rC4V were finally identified as the optimal combination of the vaccine.3.The rC4V was used as coating antigen for developing an indirect rC4V-ELISA method,and the sensitivity and specificity of the rC4V-ELISA were 84.2%and 100%,respectively.The comparative test between rC4V-ELISA and the liquid-phase blocking ELISA kit?LPB-ELISA?with 376 clinical samples showed the coincidence rate was 84.04%.This method has the potential to monitor the level of antibodies after infection and immunization.4.Four different forms of Cap-CRT fusion proteins?rP4C,rC4P,rP5F and rF5P?were successfully constructed by fusing full-length Cap proteins with different lengths of truncated CRT?120-250 aa/120-308 aa?at the N and C-terminal,respectively.The conventional optimization of soluble expression conditions successfully achieved the soluble expression of rF5P.The high purity of rF5P was obteined by Ni-NAT affinity chromatography,and rF5P was verified to form polymer by gel filtration.DLS and other methods were used to detect the diameter of rF5P was 100 nm.The virus titer of DF-1 strain was up to 10^7.5TCID₅₀/mL after continuous culture,which laying a foundation for further vaccine research.The purified rF5P vaccine was prepared with adjuvant and immunized mice.Indirect ELISA and IPMA were performed to detect the high levels of humoral immunity after immunization,and the antibody titer lasted for 56 days after immunization,which was not different from the commercial vaccines.At the same time,the lymphocyte secretion of IFN-?,IL-10,IL-18,TNF-?,GM-CSF and lymphocyte proliferation were detected.The results showed that the immunized mice could also produce a certain level of T cell immune response.After56 days of immunization,DF-1 challenged in mice,and then the virus contents in their heart,liver,spleen,lungs and kidney tissues were detected by RT-PCR.The results showed that rF5P vaccine effectively reduced the infection rate of virus in mice,and rF5P could be used as a candidate vaccine for the prevention of PCV2.