Fusion Expression And Immunogenicity Of Porcine Circovirus Type 2 Cap Protein And Recombinant Single Chain Antibody APCH1

Porcine circovirus type 2(PCV2)is one of the important pathogens that seriously endanger the pig industry in China.Currently,the prevention and control of PCV2 is mainly based on vaccine immunity.There are many commercial PCV2 vaccines on the market,most of them need to add adjuvants to improve the immune effect.Traditional adjuvants have been widely used due to their strong immune effect,but they also cause some toxic and side effects,which may cause potential health risks to the animals.Recently,studies have shown that APCH1(Antigen presenting cell homing 1)recombinant single chain antibody can carry antigen targeting to antigen presenting cell(APCs),and bind to the antigen site of major histocompatibility complex II class molecule(MHCII)forming an antigen peptide-MHCII molecular complex,and then transported to the surface of the APCs for recognition by the lymphocyte receptor(TCR),thereby significantly enhancing the immune effect of the subunit vaccine.Therefore,this study used the baculovirus expression vector system to fuse APCH1 with PCV2 Cap protein,constructed a PCV2 subunit genetic engineering vaccine with molecular adjuvant activity,and evaluated the immune efficacy of the constructed vaccine in animals.The main research contents are as follows:1.Construction and expression of the recombinant baculovirus r BV-APCH1-Cap in vitroRecombinant single-chain antibody APCH1 gene and insect cell codon optimized PCV2 ORF2 gene(encoding Cap protein)were synthesized.Using the two genes as templates,APCH1 gene and ORF2 gene(? 41ORF2)with deleted nuclear localization sequence were amplified by PCR.The recombinant transfer plasmid p Fast Bac Dual-D-APCH1-? 41ORF2 expressing APCH1-? 41ORF2 fusion gene was constructed by digestion and ligation,respectively.Subsequently,the recombinant transfer plasmid was transformed into DH10 Bac E.coli,and the positive clones were screened by blue-white spot.The recombinant r Bacmid-APCH1-? 41ORF2 genome was extracted and confirmed to have been integrated into the recombinant baculovirus Bacmid by PCR.Bacmid was transfected into Sf9 insect cells to obtain recombinant baculovirus r BV-APCH1-Cap.Indirect immunofluorescence and Western blot experiments confirmed that the recombinant baculovirus could efficiently express APCH1-Cap recombinant protein in insect cells.2.Establishment of double-antibody sandwich ELISA for quantitative detection of PCV2 Cap proteinIn order to detect the content of PCV2 Cap protein expressed by recombinant baculovirus conveniently and quickly,a recombinant baculovirus r BV-Cap-His was constructed.The expressed protein Cap-His was purified by nickel column to obtain a concentration of 0.37 mg/m L.Two specific monoclonal antibodies 2D3 and 3E10 against PCV2 Cap protein were obtained by immunizing mice with the purified protein.The monoclonal antibodies 2D3 and 3E10 were used as coating antibody and detection antibody respectively,and the purified protein was used as antigen.A double antibody sandwich ELISA method was established to detect the content of PCV2 Cap protein,and the standard curves were drawn according to the OD630 values of Cap protein with different concentration.The established ELISA method and Western blot were used to detect three purified Cap protein samples at the same time.The content of the protein detected by the two methods was determined according to the standard curve and Image J gray analysis.The results showed that there was a good correlation between the protein content obtained by the two methods,which confirmed that the double antibody sandwich ELISA method was reliable and could be used for the quantitative analysis of PCV2 Cap protein.3.Protective immunity induced by the recombinant subunit vaccine APCH1-Cap in miceTo assess the molecular adjuvant effect of APCH1,this study compared the immunogenicity of the molecular adjuvant subunit vaccine APCH1-Cap with conventional subunit vaccine Cap protein,and immunized BALB/c mice at doses of 2 ?g/mouse and 20 ?g/mouse,respectively.The results showed that the level of ELISA antibody induced by molecular adjuvant subunit vaccine APCH1-Cap and the expression of IFN-? and IL-4 were significantly higher than those of routine subunit vaccine Cap protein,and the immune response was positively correlated with the dose.The challenge experiments further showed that the neutralizing antibody induced by the molecular adjuvant subunit vaccine APCH1-Cap in mice was significantly higher than that of the routine subunit vaccine Cap protein immunization group.Histopathological results showed that a small amount of lymphocyte necrosis was observed in the spleen nodules and lymph nodes of the challenged control group,and spleen macrophages were visible.The tissue structure of mice immunized with molecular adjuvant subunit vaccine APCH1-Cap was intact and no obvious granulocyte infiltration was found.These results indicate that the molecular adjuvant APCH1 contributes to the enhancement of Cap protein-induced humoral and cellular immune responses.In summary,compared with conventional subunit vaccine,the molecular adjuvant subunit vaccine APCH1-Cap,can enhance the antigen processing and presentation of antigen presenting cells through the targeting effect of APCH1,and effectively stimulate mice to produce stronger humoral immunity and cellular immune response,so as to play a more effective immune protection.This study will lay a good experimental foundation for the research of novel and efficient porcine circovirus vaccine.In addition,the vaccine design strategy targeting MHCII molecules will also provide reference for other pathogenic microbial vaccines.